Cell Biology and Translational Medicine, Volume 20: Organ Function, Maintenance, Repair in Health and Disease (Advances in Experimental Medicine and Biology, 1436) [1st ed. 2023] 3031416872, 9783031416873

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Advances in Experimental Medicine and Biology 1436 Cell Biology and Translational Medicine

Kursad Turksen  Editor

Cell Biology and Translational Medicine, Volume 20 Organ Function, Maintenance, Repair in Health and Disease

Advances in Experimental Medicine and Biology

Cell Biology and Translational Medicine Volume 1436 Series Editor Kursad Turksen (emeritus), Ottawa Hospital Research Institute, Ottawa, ON, Canada Editorial Board Members Pascal Pineau, Institut Pasteur, Paris, France Daisuke Sugiyama, Kyushu University, Fukuoka, Japan Jeffrey M. Gimble, Louisiana State University, Baton Rouge, LA, USA Pablo Menendez, Josep Carreras Leukaemia Research Institute, Barcelona, Spain Cesar V. Borlongan, University of South Florida Health, Tampa, FL, USA Essam M. Abdelalim, Diabetes Research Institute, Doha, Qatar Aaron W. James, Johns Hopkins Hospital, Baltimore, MD, USA Srikala Raghavan, Institute for Stem Cell Science and Regenerative Medicine, Bengaluru, Karnataka, India Tiziana A. L. Brevini, University of Milan, Milan, Italy Murat Y. Elcin, Ankara University, Ankara, Türkiye Mario Tiberi, Ottawa Hospital, Ottawa, ON, Canada Nagwa El-Badri, Zewail City of Science and Technology, Giza, Egypt Panos Kouklis, University of Ioannina, Mpizani, Greece Benjamin Levi, The University of Texas Southwestern Medical Center, Dallas, TX, USA

Cell Biology and Translational Medicine aims to publish articles that integrate the current advances in Cell Biology research with the latest developments in Translational Medicine. It is the latest subseries in the highly successful Advances in Experimental Medicine and Biology book series and provides a publication vehicle for articles focusing on new developments, methods and research, as well as opinions and principles. The Series will cover both basic and applied research of the cell and its organelles’ structural and functional roles, physiology, signalling, cell stress, cell-cell communications, and its applications to the diagnosis and therapy of disease. Individual volumes may include topics covering any aspect of life sciences and biomedicine e.g. cell biology, translational medicine, stem cell research, biochemistry, biophysics, regenerative medicine, immunology, molecular biology, and genetics. However, manuscripts will be selected on the basis of their contribution and advancement of our understanding of cell biology and its advancement in translational medicine. Each volume will focus on a specific topic as selected by the Editor. All submitted manuscripts shall be reviewed by the Editor provided they are related to the theme of the volume. Accepted articles will be published online no later than two months following acceptance. The Cell Biology and Translational Medicine series is indexed in SCOPUS, Medline (PubMed), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio.

Kursad Turksen Editor

Cell Biology and Translational Medicine, Volume 20 Organ Function, Maintenance, Repair in Health and Disease

Editor Kursad Turksen (emeritus) Ottawa Hospital Research Institute Ottawa, ON, Canada

ISSN 0065-2598 ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISSN 2522-090X ISSN 2522-0918 (electronic) Cell Biology and Translational Medicine ISBN 978-3-031-41687-3 ISBN 978-3-031-41688-0 (eBook) https://doi.org/10.1007/978-3-031-41688-0 # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Preface

In this next volume in the Cell Biology and Translational Medicine series, we continue to explore how insights from state-of-the-art cell biological studies can impact translational medicine. Amongst topics explored here are recent developments that provide important new understanding of stem cell-based reprograming, and therapeutics that underscore where we stand with respect to translational medicine and novel cell- and/or drug-based therapeutic options for a variety of diseases and conditions . As with each of the volumes in this series, we continue to highlight timely, often emerging, topics and novel approaches with potential to accelerate our understanding of various diseases with the ultimate goal of improving therapeutic options. I remain very grateful to Gonzalo Cordova, the Publishing Editor of the series, and wish to acknowledge his continued support. Finally, sincere thanks to the contributors not only for their support of the series, but also for their willingness to share their insights and all their efforts to capture both the advances and the remaining obstacles in their areas of research. I trust readers will find their contributions as interesting and helpful as I have. Ottawa, ON, Canada

Kursad Turksen

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Contents

Direct Cardiac Reprogramming: Current Status and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Krishna Kumar Haridhasapavalan, Atreyee Borthakur, and Rajkumar P. Thummer

1

Stem Cell-Based Therapeutic Approaches in Genetic Diseases . . . Ayça Aslan and Selcen Arı Yuka

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Gene Therapeutic Delivery to the Salivary Glands . . . . . . . . . . . . Akshaya Upadhyay, Uyen M. N. Cao, Arvind Hariharan, Akram Almansoori, and Simon D. Tran

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Cancer Stem Cells and Their Therapeutic Usage . . . . . . . . . . . . . . Meryem Osum and Rasime Kalkan

69

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ozlem Aybuke Isik and Onur Cizmecioglu

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Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Giulia Silvani, Valentin Romanov, and Boris Martinac Drug Therapeutics Delivery to the Salivary Glands: Intraglandular and Intraductal Injections . . . . . . . . . . . . . . . . . . . 119 Akram Abdo Almansoori, Arvind Hariharan, Uyen M. N. Cao, Akshaya Upadhyay, and Simon D. Tran Negative-Pressure Wound Therapy: What We Know and What We Need to Know . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Toshifumi Yamashiro, Toshihiro Kushibiki, Yoshine Mayumi, Masato Tsuchiya, Miya Ishihara, and Ryuichi Azuma Vitamin A Deficiency, COVID-19, and Rhino-Orbital Mucormycosis (Black Fungus): An Analytical Perspective . . . . . . 153 Aziz Rodan Sarohan, Sait Edipsoy, Zeynep Gürsel Özkurt, Can Özlü, Ayça Nur Demir, and Osman Cen

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Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput Screening for Precision Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Thudzelani Takalani Austin Malise, Ekene Emmanuel Nweke, Mutsa M. Takundwa, Pascaline Fonteh Fru, and Deepak B. Thimiri Govinda Raj Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Contents

Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 1–18 https://doi.org/10.1007/5584_2022_760 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 Published online: 21 January 2023

Direct Cardiac Reprogramming: Current Status and Future Prospects Krishna Kumar Haridhasapavalan and Rajkumar P. Thummer

Abstract

Advances in cellular reprogramming articulated the path for direct cardiac lineage conversion, bypassing the pluripotent state. Direct cardiac reprogramming attracts major attention because of the low or nil regenerative ability of cardiomyocytes, resulting in permanent cell loss in various heart diseases. In the field of cardiology, balancing this loss of cardiomyocytes was highly challenging, even in the modern medical world. Soon after the discovery of cell reprogramming, direct cardiac reprogramming also became a promising alternative for heart regeneration. This review mainly focused on the various direct cardiac reprogramming approaches (integrative and non-integrative) for the derivation of induced autologous cardiomyocytes. It also explains the advancements in cardiac reprogramming over the decade with the pros and cons of each approach. Further, the review highlights the importance of clinically relevant (non-integrative) approaches and their

K. K. Haridhasapavalan, A. Borthakur, and R. P. Thummer (✉) Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India e-mail: [email protected]; [email protected]; [email protected]

, Atreyee Borthakur

,

challenges for the prospective applications for personalized medicine. Apart from direct cardiac reprogramming, it also discusses the other strategies for generating cardiomyocytes from different sources. The understanding of these strategies could pave the way for the efficient generation of integration-free functional autologous cardiomyocytes through direct cardiac reprogramming for various biomedical applications. Keywords

Cardiomyocytes · Cardiovascular diseases · Cell therapy · Direct cardiac reprogramming · Integrative and non-integrative approaches

Abbreviations 9C BMP c-Kit CVDs Dmap1 ESCs G iPSCs islet-1 LinM

9 small molecules Bone morphogenetic protein Tyrosine kinase receptors Cardiovascular diseases DNA methyltransferase 1-associated protein 1 Embryonic stem cells GATA4 Induced pluripotent stem cells Homeodomain transcription factor Lineage-negative MEF2C 1

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K. K. Haridhasapavalan et al.

PDGFRα Sca-1 T WHO

1

Platelet-derived growth factor receptor-alpha Stem cell antigen-1 TBX5 World Health Organization

Introduction

In today’s world of advancing health research, cardiovascular diseases (CVDs) still remain one of the leading causes of mortality and morbidity. The World Health Organization (WHO) in 2019 reported nearly 32% of all deceased (i.e., 17.7 million) to have succumbed to CVDs annually, which is expected to surpass 23.6 million by the next decade (Kaptoge et al. 2019). The primary CVD of concern is coronary heart disease (also known as ischemic heart disease), which accounts for 14.4% of these cases, closely followed by cerebrovascular disease, accounting for 11.2%. The major causes of CVDs are the use of tobacco, alcohol consumption, stress, poor/unhealthy diet, sedentary lifestyle, etc., in our daily life. Although ~90% of CVDs are preventable with medications, exercise, healthy diet, avoidance of tobacco, and alcohol (McGill Jr et al. 2008), an increase in mortality is observed in the recent times, which might be due to inadequate preventive measures taken against the same (Mendis et al. 2011). Out of the variety of CVDs, the ones like ischemic heart disease, cardiomyopathies and arrhythmias mostly affect the functionality of cardiomyocytes (Mendis et al. 2011), mainly due to apoptosis and necrosis in the cardiac tissue. Cardiomyocytes are the functional unit of the heart that is majorly responsible for the conduction system. In a healthy human heart, the average left ventricle has roughly 4 billion cardiomyocytes, while a post-infarct heart has a myocyte shortage of about 1 billion (Murry et al. 2006). Most of the diseases, namely, ischemic heart disease and myocardial infarction, are due to loss of regenerative capacity of the host tissue by the remaining myocytes and the consequent weakening of the diseased heart over time. The loss of cardiomyocytes leads to the formation of

scar tissue by the spontaneous division and migration of fibroblasts over the damaged area, which, in turn, results in improper contraction. This myocardial growth transition gives rise to terminally differentiated cardiomyocytes (adult) that are characterized by binucleated cells with arrested cell cycle. Naturally, human heart has a limited capacity to regenerate cardiomyocytes as indicated by lasting scar tissue following myocardial infarction and ultimately culminates in chronic heart failure in the long run. A common therapeutic approach includes pharmacotherapy, which is mainly focused on limiting disease progression instead of repairing and restoring healthy tissue and function. Therefore, the limited efficacy of this current treatment has generated interest in considering other viable and long-lasting therapeutic strategies. The search for effective ways to treat infarcted hearts has increased remarkably. In this regard, cell-based therapy for cardiac regeneration appears to be a promising alternative to achieve cardiac repair. In the near future, cell therapy could be a possible solution to control current epidemic rates of heart failure by transplanting autologous functional cardiomyocytes to support regeneration of the heart in diseased patients. Various strategies have been developed to date to make an attempt to generate cardiomyocytes from different cell types (Fig. 1), which are explained below.

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Strategies to Generate Cardiomyocytes from Different Cells

2.1

Cardiomyocytes Derived from Cardiac Progenitor Cells

Cardiac progenitor cells (also sometimes called cardiac stem cells) are a heterogeneous group of endogenous multipotent progenitor cells in the heart and are identified by the expression of various markers such as tyrosine kinase receptors (c-Kit) and/or stem cell antigen-1 (Sca-1), the homeodomain transcription factor (islet-1), platelet-derived growth factor receptor-alpha (PDGFRα), or the ability to grow into cardiospheres (Beltrami et al. 2003; Oh et al.

Direct Cardiac Reprogramming: Current Status and Future Prospects

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Fig. 1 Schematic of different strategies to generate cardiomyocytes from different sources and the biomedical application of the generated cells

2003; Messina et al. 2004; Laugwitz et al. 2005; Chong et al. 2011, 2014; Amini et al. 2017). In the year 2003, Beltrami et al. first demonstrated the differentiation ability of lineage-negative (Lin-) c-kit+ cells, isolated from the adult rat heart, toward cardiomyocytes, smooth muscle, and endothelial cells (Beltrami et al. 2003). Soon after, Schneider and colleagues established the isolation of the Sca-1+ subpopulation from adult mouse hearts and showed in vitro differentiation of these cells into cardiomyocytes in the presence of 5-azacytidine, a DNA demethylating agent (Oh et al. 2003). Notably, the authors also demonstrated the in vivo differentiation of Sca-1+ cells to cardiomyocytes after intravenous injection in mice (Oh et al. 2003). Further investigation of these cells led to the isolation of Sca-1+ and c-kit+ subpopulation of cardiosphereforming cells from both mouse and human hearts (Messina et al. 2004). Interestingly, after cardiosphere formation, spontaneously beating mouse cells were reported without co-culturing with neonatal rat cardiomyocytes, unlike the human cardiospheres, which require co-culturing of rat neonatal cells (Messina et al. 2004). Another

subpopulation expressing Isl1 was also discovered in rat, mouse, and human hearts as clusters in atria and single cells in ventricles (Laugwitz et al. 2005). These cells lack Sca-1 and c-kit expression and, when co-cultured with rat neonatal cardiomyocytes, differentiated into cardiomyocytes. Furthermore, studies have also demonstrated the derivation of cardiomyocytes from the PDGFRα+ subpopulation of cells from mouse and human hearts (Chong et al. 2011, 2014; Le and Chong 2016). Interestingly, Raghunathan et al. demonstrated the conversion of human adipogenic mesenchymal stem cells-derived cardiac progenitor cells into pacemaker-like cells, a specialized cardiomyocyte, through the ectopic expression of SHOX2, HCN2, and TBX5 transcription factors (Raghunathan et al. 2020). In general, these cardiac progenitor cells are in an inactivated or quiescent state under normal physiological conditions. In this state, cardiac progenitor cells do not contribute to the regeneration of cardiomyocytes; however, upon cardiac injury, these progenitor cells can get activated and subsequently differentiate into cardiomyocytes (Le and Chong 2016).

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Despite the identification of these different cardiac progenitor cell populations, their physiological and pathophysiological functions are not entirely understood (Amini et al. 2017). Notably, these cells induce unfavorable or regenerative effects upon exogenously delivered within the injured heart (Le and Chong 2016). Moreover, the molecular mechanisms behind these effects still remains unclear (Le and Chong 2016). Thus, the applications of these progenitor cells are limited to the regeneration of cardiac tissue after injury.

2.2

Cardiomyocytes Derived from Adult Stem Cells

Adult stem cells such as mesenchymal stem cells are another cell source that can be used to differentiate them into functional cardiomyocytes. Several studies on hematopoietic stem cells were inconclusive with respect to cardiac fate determination (Orlic et al. 2001; Balsam et al. 2004; Kawada et al. 2004; Murry et al. 2004). Therefore, researchers focused on non-hematopoietic stem cells, such as mesenchymal stem cells, as a primary source to obtain cardiomyocytes. In this regard, adult mouse bone marrow-derived non-hematopoietic stem cells developed features of cardiomyocytes when being treated with 5-azacytidine (Makino et al. 1999). Similarly, studies reported the formation of cardiomyocytes from mouse mesenchymal stem cells in the presence of 5-azacytidine (Hattan et al. 2005; Antonitsis et al. 2007) and also by injecting into the mouse embryos (Jiang et al. 2002). However, 5-azacytidine has been reported to induce carcinogenicity by introducing mutations in the somatic cell genome (Alagesan and Griffin 2014), thus serving as a roadblock to the therapeutic applications of differentiated cells. Therefore, Shen et al. focused on the downstream targets of 5-azacytidine and found the significant upregulation of miR-1-2 during differentiation (Shen et al. 2017). The authors demonstrated that mimics of miR-1-2 promoted the differentiation of bone marrow-derived mesenchymal stem cells into functional cardiomyocytes by activating

the Wnt/β-catenin signaling pathway (Shen et al. 2017). Similarly, miR-1 has been reported to induce the differentiation of mesenchymal stem cells into myocardial cells only in a specific medium, i.e., serum-free cardiomyogenic medium containing 10 nM 5-azacytidine (Zhao et al. 2016). Alternatively, Shim et al. obtained human cardiomyocyte-like cells from adult bone marrow stem cells by treating them with a low concentration (10-9 M) of dexamethasone (corticosteroid) (Shim et al. 2004). On the other hand, differentiation of bone marrow-derived clonal subpopulation by co-culturing method showed phenotypes of a heterogeneous populations of cells comprising cardiomyocytes, endothelial cells, and smooth muscle cells (Yoon et al. 2005). Likewise, Cai et al. employed the same co-culturing method with minor modifications (1:10 instead of 1:4 ratio) to differentiate bone marrow-derived mesenchymal stem cells into cardiomyocytes (Cai et al. 2012). Apart from 5-azacytidine and miRNAs, several growth factors/cytokines, microenvironment, caveolin-1, vanilloid receptor 1, and histone deacetylase 1 were reported to induce cardiac differentiation of mesenchymal stem cells (Guo et al. 2018). Moreover, these mesenchymal stem cells (along with fresh bone marrow) promoted the activation of angiogenesis, inhibition of fibrosis, and decrease in apoptosis to restore heart function in the infarcted swine model (Pak et al. 2003). Among the various sources of mesenchymal stem cells like the umbilical cord, adipose tissue, placenta, hair follicle, skeletal muscle, etc., adipose tissue serves as an easily obtainable source compared to the invasive process of bone marrow aspiration. Kakkar et al. underscore the merits associated with the induction of adipose tissue-derived stem cells with TGF-β1, which is nontoxic and a more efficient cardiac inducer compared to 5-azacytidine (Kakkar et al. 2019). Another study focused on human amniotic fluid-derived mesenchymal stem cells, which were effectively differentiated into the cardiomyogenic lineage upon treatment with 10 μM 5-azacytidine and 20% human platelet lysate (Markmee et al. 2020).

Direct Cardiac Reprogramming: Current Status and Future Prospects

Ramesh et al. summarized the use of various biological and chemical inducers that enable the cardiac differentiation of adult stem cells into cardiomyocytes (Ramesh et al. 2021). However, direct transplantation of these cells is limited due to low differentiation or success rate in vivo, and these do not entirely reciprocate the functional and morphological characteristics of cardiomyocytes (Guo et al. 2018).

2.3

Cardiomyocytes Derived from Pluripotent Stem Cells

In 1999, Guan et al. reported differentiation of undifferentiated embryonic stem cells (ESCs) into cardiomyocytes, neuronal, skeletal muscle, and epithelial and vascular smooth muscle cells. In this study, Guan et al. concluded that the differentiation of ESCs toward cardiomyocytes was influenced by cell density, medium, and its supplements, type of cells, and time of seeding cells (Guan et al. 1999). Contrastingly, Kehat et al. demonstrated cell density independent differentiation of human ESCs toward cardiomyocytes with similar structural and functional properties of early stage cardiomyocytes (Kehat et al. 2001). In this study, the authors performed differentiation by forming embryoid bodies and then seeded these embryoid bodies in 0.1% gelatin-coated petri dishes. They observed the first embryonic bodies with rhythmically contracting areas on day 4 (Kehat et al. 2001), which is 2 days earlier than the previous study (Guan et al. 1999). However, most of these studies used two different culture conditions for ESCs maintenance and differentiation. Interestingly, Denning and colleagues developed a common culture condition for the maintenance of ESCs and subsequently demonstrated efficient differentiation of these cells toward cardiomyocytes (Denning et al. 2003). Soon after the astounding discovery of induced pluripotent stem cells (iPSCs) from mouse fibroblasts (Takahashi and Yamanaka 2006), the same group generated human iPSCs from adult human fibroblasts and differentiated into cardiac and other cells as an evidence of pluripotency (Takahashi et al. 2007). For the

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directed differentiation, Yamanaka et al. followed the previously reported protocol (Laflamme et al. 2007) in which human ESCs were treated with Activin A and bone morphogenetic protein (BMP) 4 to form beating cardiomyocytes. Further, induction with Activin A and BMP4 enhanced the generation of cardiomyocytes; however, there was a lot of variability between cell lines and experiments (Paige et al. 2010). Using small molecules, Lian et al. fine-tuned the Wnt/β-catenin signaling pathway to generate cardiomyocytes robustly from multiple pluripotent cells. In this study, the authors showed that Wnt signaling activation is crucial for mesoderm formation from pluripotent cells, whereas its inhibition was crucial for the sequential differentiation of these cells into cardiomyocytes (Lian et al. 2012). This was further fine-tuned by Kadari et al. which reported the formation of three different phases, namely, cardiovascular induction, cardiac specification, and cardiomyocyte enrichment (Kadari et al. 2015). In the first phase, authors used CHIR99021 and BMP4 to stimulate cardiac induction and then used Wnt inhibitor (XAV939) to induce cardiac specification (Kadari et al. 2015). In order to reduce line-to-line variability, they performed lactate enrichment to obtain pure populations of cardiomyocytes with a very high efficiency (Kadari et al. 2015). Ou et al. described a protocol wherein co-culturing of iPSCs with neonatal cardiomyocytes resulted in cardiomyocytes expressing cardiac-specific genes like Mef2c, cTnT, and MLC-2 V (Ou et al. 2016), indicating efficient differentiation and enhanced proliferation ability upon co-culture. Studies also suggest the administration of electrical stimulations in human iPSC or cardiosphere-derived cells to achieve functionally mature cardiomyocytes (Ma et al. 2018; Nazari et al. 2020). These stimulations could mimic the native property of synchronous contractions of the heart and aid in attaining a functionally mature state in shorter time duration (Ma et al. 2018; Nazari et al. 2020). Funakoshi et al. provided insights into the advantage of generating human PSC-derived mature cardiomyocytes that have enhanced contraction force, mitochondrial oxidative property, and improved sarcomere structure (Funakoshi et al.

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2021). Transplantation of these mature cells compared to the functionally immature ones generated better grafts, suggesting the prior manipulation of the cells before the transplant could help mitigate arrhythmias and serve as a safer therapy (Funakoshi et al. 2021). Exciting opportunities exist in the field of PSC-derived cardiomyocytes, but not without a series of hurdles preventing its use in the clinical setting. Some of them include the fetal-like immature phenotype of generated cardiomyocytes, variability in the cardiac subtype, the risk of arrhythmogenesis upon transplant, teratoma formation, and immune rejection, to name a few.

2.4

Direct Cardiac Reprogramming Strategies to Derive Cardiomyocytes

2.4.1

Direct Cardiac Reprogramming of Mouse Fibroblasts Several studies have generated induced cardiomyocytes by directly reprogramming somatic cells using different integrative and non-integrative (Fig. 2; Table 1) approaches

Fig. 2 Schematic illustration of different direct cardiac reprogramming approaches. FSP1, fibroblast specific protein 1; Col1a1, collagen alpha-1(I) chain; Col1a2, collagen alpha-2(I) chain; DDR2, discoidin domain receptor 2;

(Ieda et al. 2010; Fu et al. 2013; Nam et al. 2013; Wada et al. 2013; Muraoka et al. 2014; Wang et al. 2014, 2015 Lee et al. 2015; Cao et al. 2016; Miyamoto et al. 2018; Paoletti et al. 2020; Yamakawa and Ieda 2021). The very first study to generate fibroblast-derived induced cardiomyocytes was reported in 2010 (Ieda et al. 2010). The authors selected 13 potential cardiogenic factors critical for survival and cardiogenesis in the embryo from the previously reported microarray analysis data of variable expression patterns observed between myocytes and non-myocytes cells (Ieda et al. 2009). Additionally, mesoderm-specific transcription factor-1 (Mesp1) was also included due to its cardiac reprogramming ability in Xenopus laevis (David et al. 2008). Of these 14 factors, this study identified a combination of three transcription factors, namely, GATA4, MEF2C, and TBX5 (referred to as GMT), sufficient to reprogram mouse cardiac/dermal fibroblasts to induced cardiomyocytes (Ieda et al. 2010), bypassing the pluripotent stem cell state. These induced cells exhibited almost similar gene expression pattern and electrophysiology and contracted spontaneously as native cardiomyocytes. However, the

cTnT, cardiac Troponin T; Cx43, connexin 43; NKX2.5, NK2 Homeobox 5; MLC2v/2a, myosin light chain-2cardiac ventricular/atrial isoforms

Direct Cardiac Reprogramming: Current Status and Future Prospects

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Table 1 Overview of reprogramming approaches (mouse) Reprogramming approaches Retroviral vectors

Reprogramming factors GMT

Retroviral vectors Lentiviral vectors Retroviral vectors Lentiviral vectors Lentiviral vectors Retroviral vectors Retroviral vectors Lentiviral vectors

OSKM

Source cell Postnatal cardiac/ dermal fibroblasts MEF

TM + MyoCD

MEF

GMTH

TTF, CF

miR-1, 133, 208, 499 + JAK inhibitor I HNGMT

Noncardiac myocytes MEF

GHMT

TTF

GMT + miR-133a

MEF

HNGMT + SB431542

MEF, ACF

OCT4 + Parnate, Forskolin, CHIR99021, SB431542 OSKM + vitamin C

MEF, TTF

GHMT + miR-1, miR-133 + Y-27632, A83–01 GMT

MEF, AF

GMT + BMI1 deletion GMT

TTF, CF, MEF RCF

GMT

RCF

GMT + SB431542, XAV939 GHMT + AKT, ZNF281 GMTH

MCF

Lentiviral vectors

Retroviral vectors Retroviral vectors mRNA Retroviral and lentiviral vectors Lentiviral vectors Adenoviral vectors Retroviral vectors Retroviral vectors Lentiviral vectors Adenoassociated virus (AAV) Lentiviral vectors

MEF

NCF

TTF TTF

AAV-GMT and AAV-Tβ4 (chimeric)

MEF, MCF

GMT/GMTHMyoCD

p63 knockout MEF

Cardiac gene expression 30% GFP+ cells, beating CMs

40% cTnT+ cells, spontaneously contracting, beating CMs 2% MYH6: tdTomato+ cells, 12% cTnT+ cells 9.5% more CMs than GMT Tenfold increased efficiency of reprogramming >50-fold efficient than GMT alone 2% cTnT+ cells, increased to 17% with MyoCD Sevenfold increased efficiency than GMT Fivefold increased efficiency of iCM generation compared to GMT (2% vs. 0.25%) Spontaneously contracting iCMs

In vivo Yes

No No Yes Yes Yes No No Yes

References Ieda et al. (2010)

Efe et al. (2011) Protze et al. (2012) Song et al. (2012) Jayawardena et al. (2012) Addis et al. (2013) Nam et al. (2013) Muraoka et al. (2014) Ifkovits et al. (2014)

No

Wang et al. (2014)

Enhanced expression and beating cardiomyocytes (day 11) 60% enhanced efficiency, contracting iCMs in 4 weeks 72.4% α-MHC+ iCMs, cardiac markers (α-MHC, β-MHC, ANF, N, and cTnT) cTnT expression highest in the GMTTβ4 group compared to GMT (32% vs. 22%) 20-fold cTnT expression, three– fivefold GMT expression, spontaneous contractions in co-culture

No

No

No Yes Yes Yes No No

Lee et al. (2015) Zhou et al. (2016) Mathison et al. (2017) Mathison et al. (2017) Mohamed et al. (2017) Zhou et al. (2017) Tian et al. (2018)

Yes

Yoo et al. (2018)

No

Patel et al. (2018)

(continued)

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K. K. Haridhasapavalan et al.

Table 1 (continued) Reprogramming approaches Sendai viral vectors

Reprogramming factors GMT

Source cell MEF, TTF

Retroviral vectors Retroviral vectors (quadcistronic) Retroviral vectors

GMTH

MEF

GMTH

MEF

GMT + IGF-1, MM589, A83-01, PTC-209

MEF, NCF

Retroviral vectors Lentiviral vectors

GT + M isoform (Mi2/Mi4) GMT + sodium butyrate, ICG-001, retinoic acid CHIR99021, RepSox, Forskolin, valproic acid, Parnate, TTNPB + PTC-209 GMT

MEF

Small molecules

Sendai viral vectors Adenoviral vectors Retroviral and lentiviral vectors Retroviral vectors Retroviral vectors

OSKM

Cardiac gene expression Sendai viral-GMT vectors more efficient than retroviral-GMT vectors, 20% cTnT+ cells were matured during in vivo cardiac reprogramming iCMs with enhanced contractile cardiac structures Splicing order of M-G-T-Henhanced reprogramming

In vivo Yes

No No

References Miyamoto et al. (2018)

Zhang et al. (2019) Zhang et al. (2019)

Three–fourfold increase in cTnT and α-actinin (MEF); eight– ninefold increase in spontaneous beating of iCMs (NCF) Efficiency of Mi2 > Mi4 isoform

No

Guo et al. (2019)

No

RCF

4-fold cTnT expression, spontaneous contractions >4 week

No

Wang et al. (2020) Singh et al. (2020)

MEF, CF

α-MHC+ cTnT+, spontaneously beating iCMs

No

Testa et al. (2020)

MCF

iCMs (tdTomato and cTnT+) > > 4 weeks Partial in vivo reprogramming, CDH1+, FUT4+ cells

Yes

Isomi et al. (2021) Kisby et al. (2021)

GMTMyoCD, Sall4

Myocardial infarction model MICF

GMT + SB431542

TTF, MEF

GHMT + DMSO

MEF

Beating iCMs >28 day, 22.5% cTnT+ cells ~twofold more iCMs compared to control ~threefold cTnT+ cells, ~sixfold increase in MYH6-mCherry+ cells

Yes

No No No

Zhao et al. (2021) Bektik et al. (2021) Lim et al. (2021)

G-GATA4; M, MEF2C; T, TBX5; H, HAND2; N, NKX 2.5; HNGMT, Hand2; Nkx 2.5 + GMT; OSKM, Oct3/4,Sox2,Klf4, Myc; MyoCD, myocardin; miRNA, microRNA; JAK, janus kinase; CM, cardiomyocyte; iCM, induced CM; GFP, green fluorescent protein; MEF, mouse embryonic fibroblasts; CF, cardiac fibroblasts; TTF, tail tip fibroblasts; ACF, adult cardiac fibroblasts; AF, adult fibroblasts; NCF, neonatal cardiac fibroblasts; RCF, rat cardiac fibroblasts; MICF, cultured cardiac fibroblasts isolated from adult mice with myocardial infarction; cTnT, cardiac troponin T; MYH/MHC, myosin heavy chain; ANF, natriuretic peptide A; CDH1, cadherin-1; FUT4, fucosyltransferase 4; AAV-Tβ4, adeno-associated virus carrying thymosin Β4; BMI1, polycomb complex protein; AKT, protein kinase B; ZNF281, zinc finger protein 281; TGFβ, transforming growth factor beta; DMSO, dimethyl sulfoxide

efficiency of the beating cells was very low, and the majority of the cell population was only partially reprogrammed. The reason behind this inefficient reprogramming is that the likelihood of transducing a single cell with all three factors is low and the imbalanced stoichiometric expression of these factors (Lee et al. 2015; Wang et al. 2015). Relatively high levels of MEF2C protein

expression compared to GATA4 and TBX5 improved reprogramming efficiency and the quality of induced cardiomyocytes (Wang et al. 2015). Of the two isomeric forms of MEF2C, viz., Mi2 and Mi4, the former, in combination with GATA4 and TBX5, reprogrammed mouse embryonic fibroblasts more efficiently than the latter (Wang et al. 2015). This might be

Direct Cardiac Reprogramming: Current Status and Future Prospects

the possible reason for the discrepancy in reprogramming efficiency reported by different groups (Yamakawa and Ieda 2021). Further, the addition of HAND2 to this reprogramming cocktail enhanced the efficiency, irrespective of the stoichiometry using either cocktail of retroviralGMT vectors or retroviral single polycistronicMGT vector (Song et al. 2012; Nam et al. 2013; Zhang et al. 2019; Wang et al. 2020b). These studies thus demonstrated the requirement of HAND2 in the GMT cocktail (GMT + HAND2) in the direct cardiac reprogramming of mouse fibroblasts into functional cardiomyocytes. Using the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC), Efe et al. partially reprogrammed mouse fibroblasts, instead of inducing full pluripotency, and then derived cardiomyocytes by diverting them toward cardiac lineage with specific media conditions (Efe et al. 2011). The first spontaneous beating was observed after 11 days in this approach (Efe et al. 2011), compared to 4–5 weeks in the first study (Ieda et al. 2010). The addition of ascorbic acid (Vitamin C) to the Yamanaka factors enhances the derivation of cardiomyocytes from mouse fibroblasts through a partial pluripotent reprogramming strategy (Talkhabi et al. 2015). With a different set of transcription factors (TBX5, MEF2C, and MYOCD), cardiomyocytelike cells were obtained by time-dependent conversion of mouse embryonic fibroblasts through the lentiviral expression of these three factors, upregulating a broader spectrum of cardiac genes (Protze et al. 2012), compared to the combination used by Ieda et al. (2010). Similarly, screening a combination of transcription factors to establish a minimally efficient reprogramming cocktail, Hirai et al. reported the use of MYOCD with MEF2C and GATA6 to generate smooth muscle resembling cells with characteristic cardiac marker genes and reduced fibroblast-specific gene expression (Hirai et al. 2018). Delivering miRNAs in an integration-free manner, Jayawardena et al. established the derivation of cells having characteristics of cardiomyocytes via transient expression of muscle-specific miR-1, miR-133, miR-208, and miR-499 in mouse cardiac fibroblasts by a single

9

transfection (Jayawardena et al. 2012). The authors also reported further enhancement of cardiomyocyte-like cells by the addition of JAK inhibitor I, which is believed to induce expression of cardiac ion channels as well as enhance α-MHC (Jayawardena et al. 2012). Following this, Muraoka et al. included miR-133 in the GMT reprogramming cocktail and demonstrated a sevenfold increase in the reprogramming efficiency and improved kinetics of 99% accuracy and specificity. In the future, it was claimed that this will provide an opportunity to develop advanced real-time epigenomics monitoring technologies such as nucleus-targeted drug monitoring and epigenomic prognosis and diagnostics (Verma et al. 2022).

81

5

Conclusion

CSCs play a crucial role in invasion, metastasis, therapeutic resistance, and cancer relapse. Therefore, nowadays, the identification and targeting of various mechanisms regulating CSC properties have begun to attract more attention. Most antiCSC therapeutic strategies target stemness-related factors and pathways shared between CSCs and normal SCs, leading to off-target effects. Therefore, the development of novel therapeutic strategies specifically targeting CSCs is essential. It was suggested that novel therapeutic strategies for elimination of CSC populations should contain multiple drugs targeting ABC transporters, DNA damage repair mechanisms, tumor microenvironment, EMT, antitumor immunity, and stemness-related pathways. Considering CD44 variant (CD44v) isoform was found to have high tumor initiation capacity, and CSC heterogeneity, the identification, and targeting of multiple CSC-specific antigens expressed on various tumors will be an effective strategy to eliminate CSCs. Nanoparticles have been proposed as important tools for targeting resistance mechanisms in CSCs due to their ability to target chemotherapeutic drugs at higher concentrations to target sites and CSCs. Choosing the least toxic and immunogenic nanoparticles is crucial for effective cancer treatment. Due to tumor migration and low immunogenicity of MSCs, they can also be used as a delivery vehicle to provide effective cancer treatment. Consequently, the use of drug or factor-loaded NPs or MSCs modified with multiple antibodies/factors targeting concurrently multiple antigens/factors on CSCs can significantly decrease drug toxicity and side effects and increase the effectiveness of cancer treatment. Therefore, it is thought that focusing on studies in this area will provide great advantages in terms of cancer treatment in the future.

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Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 87–108 https://doi.org/10.1007/5584_2022_759 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 Published online: 18 January 2023

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease Ozlem Aybuke Isik and Onur Cizmecioglu

Abstract

Keywords

The plasma membrane is not a uniform phospholipid bilayer; it has specialized membrane nano- or microdomains called lipid rafts. Lipid rafts are small cholesterol and sphingolipidrich plasma membrane islands. Although their existence was long debated, their presence in the plasma membrane of living cells is now well accepted with the advent of superresolution imaging techniques. It is interesting to note that lipid rafts function to compartmentalize receptors and their regulators and substantially modulate cellular signaling. In this review, we will examine the role of lipid rafts and caveolae-lipid raft-like microdomains with a distinct 3D morphology—in cellular signaling. Moreover, we will investigate how raft compartmentalized signaling regulates diverse physiological processes such as proliferation, apoptosis, immune signaling, and development. Also, the deregulation of lipid raft-mediated signaling during tumorigenesis and metastasis will be explored.

Cancer · Caveolae · Lipid rafts · Plasma membrane compartmentalization · Signal transduction

Abbreviations CAF Cav CBM CSD DISC DRM DSM ECM EGF Signaling EMT FADD GPI-anchor IGF Signaling IRS-1

O. A. Isik and O. Cizmecioglu (✉) Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey e-mail: [email protected]

LAT Ld Lo

Cancer-associating fibroblasts Caveolin Caveolin binding motif Caveolin scaffolding domain Death-inducing signaling complex Detergent-resistant membranes Detergent-soluble membranes Extracellular matrix Epidermal growth factor signaling Epithelial-mesenchymal transition Fas-associated death domain Glycosylphosphatidylinositol anchor Insulin-like growth factor signaling Insulin receptor substrate protein 1 Linker for activation of T cells Liquid disordered Liquid ordered 87

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MAPK Signaling MβCD NSOM PALM PI3K PM PTEN STED Microscopy STORM TEM

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Mitogen-activated protein kinase signaling Methyl-β-cyclodextrin Near-field scanning optical Photoactivated localization microscopy Phosphatidylinositol-3 kinase Plasma membrane Phosphatase and tensin homolog Stimulated emission depletion microscopy Stochastic optical reconstruction microscopy Tetraspanin-enriched membrane rafts

The Compartments of the Plasma Membrane as Organizers of Cellular Signaling

Plasma membrane (PM) has long been viewed as a homogenous phospholipid bilayer dispersed randomly with membrane-bound proteins (Singer and Nicolson 1972). This view was challenged by identification of many subcompartments of the plasma membrane with various compaction properties and protein composition (Yu et al. 1973). These subcompartments of PM—so-called lipid rafts—are laterally mobile, enriched in cholesterol and saturated sphingolipids. They are now recognized for their crucial roles as platforms recruiting regulatory signaling molecules and facilitating effective signal transduction.

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Characterization of Lipid Rafts

Biochemical experiments initially revealed that extracting PM at cold temperatures by nonionic detergents, such as Triton X-100, yields two populations of PM lipids (Yu et al. 1973). These are named detergent-soluble membranes (DSMs) and detergent-resistant membranes (DRMs) (Bagatolli and Mouritsen 2013). This observation put forth the idea that the lipidic composition of

PM is heterogenous rather than being a homogenous phospholipid bilayer as previously assumed (Ahmed et al. 1997; Brown and Rose 1992; Friedrichson and Kurzchalia 1998; Pralle et al. 2000; van Meer et al. 1987; Varma and Mayor 1998). Nevertheless, these findings were also challenged, as the protein composition of DRMs exhibits alterations depending on the detergent type and the detergent concentration used in lipid extraction (Mayor and Maxfield 1995; Schuck et al. 2003). The heterogeneous nature of PM lipids was explored and shown complementarily in model membranes (Feigenson and Buboltz 2001; McConnell et al. 1984; Tamm and McConnell 1985; Veatch and Keller 2003). Model membranes exhibit a phenomenon called liquid–liquid phase separation in which lipidic molecules spontaneously separate into Liquid ordered (Lo) (Hjort Ipsen et al. 1987) and Liquid disordered (Ld) (Kaiser et al. 2009; Veatch and Keller 2003) compartments. Interestingly, both Lo membranes and DRMs exhibit enrichment in terms of cholesterol and glycosylated lipids, lending credence to the lipid raft hypothesis. However, studying lipid rafts in model membranes has its handicaps. For example, sharp phase separation between compartments was observed in model membranes, yet in natural membranes, it is believed that phase separation exists as a more dynamic gradient (Sezgin et al. 2012). Also, the percentage of protein molecules incorporated into model membranes falls short compared to the protein percentage in natural membranes, which is around 25% (Dupuy and Engelman 2008). Therefore, most complex regulatory processes occurring during raft formation and cellular signal regulation through rafts are hard to examine in model membranes. More recently, a plethora of fluorescent microscopy, electron microscopy, and label-free spectroscopy techniques have been developed to study lipid rafts. And all were presented with their advantages and disadvantages (Sezgin and Schwille 2011). However, with the advancement in superresolution microscopy, the resolution limit of traditional fluorescence microscopy, which is

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease

200 nm, has been overcome (Hell and Wichmann 1994; Klar et al. 2001). As a result, we are now able to resolve objects smaller than 200 nm and conduct in-depth research on lipid rafts. Stimulated emission depletion (STED) (Hell and Wichmann 1994; Klar et al. 2001), photoactivated localization microscopy (PALM) (Sengupta et al. 2011; Hess et al. 2006; Rust et al. 2006), stochastic optical reconstruction microscopy (STORM) (Rust et al. 2006), and near-field scanning optical (NSOM) (Sezgin 2017) are some of the superresolution microscopy methodologies that are utilized in order to study PM biology and composition (you may refer to Sezgin 2017, for a more detailed review on the nuances between superresolution microscopy techniques) (Sezgin 2017). For example, STED is used to characterize the tetraspanin-enriched membrane rafts (TEMs), a high-order organization of membranous tetraspanins (Zuidscherwoude et al. 2015). Before, all proteins associated with TEMs were believed to localize to the same raft compartment. Nonetheless, this study established the existence of several different types of TEMs (Zuidscherwoude et al. 2015). Similarly, using the PALM technique, it was shown that raft-resident T-cell receptor and LAT protein locate in different raft compartments in T lymphocytes (Lillemeier et al. 2010; Rossy et al. 2013; Sherman et al. 2011). Furthermore, the STORM and PALM techniques revealed the segmented arrangement of B lymphocyte and mast cell receptors in the PM (Dustin et al. 2017; Shelby et al. 2013). Additionally, NSOM reveals raft compositions in artificial membranes (Burgos et al. 2003; Coban et al. 2007; Hollars and Dunn 1997; Hwang et al. 1998). All in all, the development of these highly sensitive microscopy tools sheds light on the previously unknown life of lipid rafts and promises more exciting times for cellular biologists. So, what exactly are “lipid rafts”? According to the Keystone Symposia on lipid rafts in 2006, lipid rafts are PM microdomains enriched in cholesterol, sphingolipids, and glycosylated lipids and are no larger than 200 nm. Furthermore, lipid rafts can recruit smaller rafts to construct larger platform-like PM domains, which constitute complex signaling hubs within PM (Pralle

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et al. 2000). Lipid rafts are separated into planar lipid rafts and flask-shaped non-planar lipid rafts (caveolae) (Ludwig et al. 2016; Simons and Ikonen 1997; Yamada 1955). Planar lipid rafts exist in all cell types and are marked by the presence of Flotillin protein (Babuke and Tikkanen 2007; Staubach and Hanisch 2011; Stuermer 2010, 2011). Caveolae is present only in specific tissues like endothelium and marked by Caveolin and Cavin proteins (Hill et al. 2008; Kurzchalia et al. 1992; Rothberg et al. 1992; Way and Parton 1995). Although they differ in morphology and chemical content, both lipid rafts have been shown to significantly modulate cellular signaling. In this review, the term “lipid raft” will be used for planar lipid rafts. In the following section, the role of lipid rafts in cellular signaling will be explained in different physiological contexts. In the last section of this review, caveolae and the role of those tiny pits in cellular signaling will be reviewed.

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Lipid Rafts in Normal Physiology

The compartmentalization of the members of the cellular signaling machinery is crucial for receiving a specific signal and generating a specific response—the goal of signal transduction. Lipid rafts are one of the tools that are utilized by cells to spatially regulate the signal. Membrane proteins are targeted to PM through their membrane-spanning and hydrophobic transmembrane domains, glycosylphosphatidylinositol (GPI)-anchors, and the covalent attachment of lipid moieties (Levental et al. 2010a; Resh 2013). Among them, GPI anchors and a subset of lipid modifications are associated with targeting proteins further in lipid rafts (Levental et al. 2010a). For example, S-palmitoylation of proteins is a well-characterized lipid modification to target proteins into rafts. The long-saturatedfatty acyl chain of palmitate (with 16 carbon atoms) is hydrophobic enough and, therefore, can integrate into lipid rafts. Also, palmitoylation is a reversible PTM, which gives targeted proteins flexibility to move in or out of the lipid rafts

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(Levental et al. 2010b). Besides palmitoylation, dual acylation of proteins (consecutively adding two lipid acyl moieties) is simultaneously associated with raft-targeting, such as myristate and isoprenoid groups. However, none of those modifications alone is sufficient for lipid raft compartmentalization (Levental et al. 2010a; Resh 2013). The myristate acyl lacks the required hydrophobicity, and isoprenoid groups have unsaturated acyl chains. Another strategy used to target proteins in the rafts is the addition of cholesterol (Porter et al. 1996). Upon secretion into the extracellular space, Hedgehog proteins are directed to PM of adjacent cells in this manner (Gallet et al. 2006; Porter et al. 1996). Once inside the lipid rafts, receptors interact with their activators or inhibitors (Simons and Toomre 2000). Hence, the molecular processes taking place in the raft or non-raft portions of the plasma membrane are of paramount significance. One other way lipid rafts might affect signaling is that the folding and therefore the functions of PM resident proteins can be altered by specific lipidprotein interactions (Laganowsky et al. 2014; Lingwood et al. 2011). Although these are two general modes of regulation in lipid rafts, they have been specifically implicated in many signaling cascades involving proliferation, apoptosis, immune responses, and development (Fig. 1).

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Proliferation

Cellular proliferation is primarily sustained by growth factor signaling through the receptor tyrosine kinases. The activities of many receptor tyrosine kinases (RTKs) are regulated through lipid rafts (Pike 2005; Simons and Toomre 2000). Epidermal growth factor (EGF) and insulin-like growth factor (IGF) are two examples of receptor tyrosine kinases associated with lipid rafts (Simons and Toomre 2000). These receptors activate effectors such as Ras-MAPK and PI3K/Akt to sustain the proliferative state (Pollak 2008; Wee and Wang 2017). EGFR is the most studied receptor tyrosine kinase in terms of its mode of activation. For the activation of downstream signaling components, EGFR molecules must be

oligomerized and autophosphorylated. Both of these activities are demonstrated to be controlled by lipid rafts (Coskun et al. 2011; Kovacs et al. 2022). Many studies have shown that disruption of lipid rafts by a cholesterol-depleting reagent— MβCD (Methyl-β-cyclodextrin)—causes aberrant activation of EGFR in the presence or absence of EGF (Jans et al. 2004; Pike et al. 2005; Roepstorff et al. 2002; Waugh et al. 1999). Therefore, to be activated appropriately, EGFR first needs to be present in raft regions; then, it needs to move to the non-raft plasma membrane after being fully activated (Kovacs et al. 2022; Lambert et al. 2006; Roepstorff et al. 2002; Turk et al. 2012; Waugh et al. 1999). Several raft-dependent regulatory mechanisms are described for IGF signaling as well. IGF signaling is initiated by insulin or IGF binding to the insulin receptor or IGFR, which results in receptor phosphorylation and activation. In 3T3-L1 adipocytes, IGFR1 is demonstrated to localize in lipid rafts (Hong et al. 2004; Huo et al. 2003). However, unlike EGFR, cholesterol depletion causes reduced IGF signal, and IGFR seems to be active only in raft regions (Huo et al. 2003). After IGFR activation, the primary effector phosphatidylinositol-3 kinase (PI3K) gets recruited to phosphorylated IGFR (therefore to PM). This recruitment occurs through the SH2 domain of the negative regulatory subunit of the PI3K-p85. On the PM, PI3K is activated after binding to IGFR (Alessi et al. 1997). Activated PI3K phosphorylates the membrane lipid PIP2 and generates a lipidic secondary messenger PIP3. Transiently produced PIP3 is recognized by the PH domain, which is present within many proteins/kinases involved in cellular proliferation (Ebner et al. 2017). AKT is one of these proteins recruited to PM by its PH domain (Brazil and Hemmings 2001; Ebner et al. 2017). Upon recruitment to the PM, AKT is activated by two sequential phosphorylation events, initiates many pathways of ultimate proliferation, and inhibits apoptosis (Manning and Cantley 2007; Manning and Toker 2017). It is interesting to note that the rafts contribute to the AKT activation. Through microscopy-based methods, active AKT was demonstrated in lipid

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Fig. 1 Modes of signaling regulation related to lipid rafts. Receptors are potentiated within the lipid rafts. The immature association of receptors with non-raft PM causes aberrant overactivation, as in the case of EGFR (I). Both receptors and coactivators are localized to different species of lipid rafts and get associated within the same lipid raft as in the case of TCR and FcɛRI signaling (II). Receptors move to non-raft PM and are proteolytically cleaved, leading EMT as in the case of CD44 signaling (III).

Lipid raft-mediated differential localization of activators and inhibitors compartmentalizes signal transduction as in the case of PI3K signaling (IV). Accumulation of signaling complexes to initiate an intracellular response, as in the case of CD95/FasL signaling(V). Caveolar signal regulation occurs through caveolar coat protein Cav 1, which binds to many signaling molecules and establishes molecular machinery (VI)

rafts, and complementary studies revealed that cholesterol depletion ablates AKT activity (Gao et al. 2011; Lasserre et al. 2008). PI3K activity is

reversed by phosphatase and tensin homolog (PTEN), which dephosphorylates PIP3 lipids into PIP2 (Song et al. 2012; Stambolic et al.

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1998). Interestingly, PTEN, the negative regulator of AKT activation, has been shown to localize in non-raft membranes, whereas PDK1, the activator of AKT, has been shown to localize in raft regions. Of note, targeting PTEN to lipid rafts through molecular methods leads to PDK1 inhibition and abrogates AKT activation (Gao et al. 2011). PI3Ks have four isoforms, two of which are ubiquitously expressed (Fruman et al. 2017). However, these two isoforms—namely PI3Kα and PI3Kβ—have differential activities and regulations in cell signaling (Jia et al. 2008a, 2008b; Vanhaesebroeck et al. 2010; Zhao et al. 2006). For example, PI3Kα is the main effector and gets activated by RTK signaling (Jia et al. 2008a, 2008b; Zhao et al. 2006). On the other hand, PI3Kβ was found to signal through GPCRs (Jia et al. 2008a, 2008b). This was found to be mediated via lipid rafts. PI3Kα localizes in non-raft areas, whereas PI3Kβ interacts with Rac1 to localize to lipid rafts. This raft localization of PI3Kβ is crucial for GPCR coupling and appears to be important in a PTEN-null genetic background (Cizmecioglu et al. 2016) (Fig. 2). The uptake of nutrients is also associated with the PI3K axis and lipid rafts. For example, in 3T3 adipocytes, when the IGF pathway is inactive,

extracellular glucose transporter GluT4 is constitutively internalized. However, once activated, IGF signaling inhibits the internalization of GluT4. Uninternalized GluT4 gets colocalized with raft-resident protein flotillin or caveolaeresident protein caveolin3 and mediates glucose uptake (Cav-3) (Fecchi et al. 2006; Ribon et al. 2001). Also, long-chain fatty acid uptake can be regulated through lipid rafts. Fatty acid translocase (FAT)/CD36 was demonstrated to be extracted from the DRM fraction of PM but not from the DSM fraction (Pohl et al. 2004, 2005). Other than receptor tyrosine kinases, some G-protein coupled receptors (GPCRs) were involved in raft-associated signaling (Ostrom and Insel 2004). Beta-adrenergic, oxytocin, serotonin, and dopamine receptors are among the GPCRs that regulate normal physiological states in the muscular and nervous systems (Björk et al. 2010; Chini and Parenti 2004; Cole and Sood 2012; Ostrom and Insel 2004). Also, several ligand-gated ion channels such as ionotropic receptors are associated with lipid rafts of neurons and muscle cells. Several glutamate receptors and GABA receptors are among them, which were demonstrated to localize to lipid rafts to relay proper excitatory signals in their target tissue

Fig. 2 Regulation of isoform-specific PI3K activity through lipid raft-based compartmentalization. Under resting conditions, PI3Kα localizes to non-raft PM, while PI3Kβ localizes to raft PM together with Gβγ and Rac1, promoting GPCR signaling (I). In PTEN wt cellular

conditions, non-raft PM localized PTEN blocks propagation of PIP3 production and signal (II). The blockage of PIP3 production in non-raft PM is compromised in PTENnull cells, and raft-associated GPCR/PI3Kβ signaling gets amplified (III)

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease

(Chini and Parenti 2004; Dalskov et al. 2005; Xu et al. 2014). Aside from GPCRs, membrane compartmentalization has been shown to modulate their related heterotrimeric G proteins (Moffett et al. 2000). Gβγ subunit of heterotrimeric G associates mostly with non-raft PM, while Gα associates with lipid rafts or caveolae. Interestingly, Gα subtypes—Gαq, Gαi, Gαs—also localize differentially to PM compartments. Gαi and Gαs localize to lipid rafts, while Gαq localizes to caveolae through its interactions with Caveolin 1 (a caveolae-resident protein) (Oh and Schnitzer 2001). Gq, on the other hand, has been shown to localize to planar lipid rafts via its interaction with the raft marker flotillin, which stimulates proliferation via the p38/MAPK axis (Sugawara et al. 2007).

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Apoptosis

Apoptosis, or “programmed cell death,” is a physiological process by which cells die during development to give rise to normal morphology or during the cell cycle when cells cannot pass the cell cycle checkpoint due to an acquired mutation. During type I apoptosis, the raft-resident CD95 (Fas) receptor needs to be bound by FasL. As a TNF family protein, CD95 possesses a cytosolic Death Domain (DD), yet the DD of CD95 functions to accumulate CD95 through protein interactions rather than induce apoptosis by itself (Ferrao and Wu 2012; Mollinedo and Gajate 2006). Liganded CD95 needs to accumulate in PM. In time, if apoptotic signals increase, accumulation of liganded CD95 reaches a threshold and recruits Fas-Associated Death Domain (FADD) (Kischkel et al. 1995; Tibbetts et al. 2003) and consecutively Caspase -8/-10 through FADD. With the recruitment of caspases, Fas/CD95, FADD, and Caspase -8/-10 form the deathinducing signaling complex (DISC) (Kischkel et al. 1995). Accumulated procaspase-8 self-cleavage occurs and initiates apoptotic signaling (Muzio et al. 1998). Following ligand interaction, CD95 travels to lipid rafts where its accumulation takes place, leading to the successful production of an apoptotic signal (Hueber et al. 2002). When

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cells are treated with cholesterol-depleting MβCD, it was demonstrated that apoptosis is inhibited because of the prevention of lipid raft formation, accumulation of CD95, and formation of DISC (Gajate et al. 2009; Gajate and Mollinedo 2015; Hueber et al. 2002; Wajant 2006).

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Immune Signaling

Lipid rafts are involved in many immunogenic processes. Firstly, one of the members of the innate immune system—Toll-like receptor (TLR4), is shown to be activated by raft-resident protein-CD14 (Wright et al. 1990). CD14 is known for its role in the pathogen recognition process. For this purpose, it forms a ternary complex with bacterial lipopolysaccharide (LPS) and the host LPS binding protein to initiate the immune response (Płóciennikowska et al. 2015; Triantafilou et al. 2011; Wright et al. 1990). It is also worth mentioning that several TLRs have cholesterol-binding motifs, and cholesterol is found to regulate the responses of those TLRs (Wong et al. 2009). Aside from innate immunity, IgE signaling in mast cells is the first known lipid raft-dependent signaling. IgE ligand binds to its receptor FcɛRI (Field et al. 1995). Once bound and activated, they go to DRMs to initiate the appropriate signaling (Field et al. 1995). Lymphocyte maturation appears to be through lipid rafts as well. In resting state, BCR and TCR are localized into DSM but once activated; they transfer into DRMs (Beck-García et al. 2015; Dustin et al. 2017; Sproul et al. 2000). Upon their transfer to DRMs, these receptors can colocalize with raft-resident downstream signaling elements of immune signaling such as LAT, LCK, and FYN kinases and start the immune response (Filipp et al. 2004; Levental et al. 2010b).

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Lipid Rafts in Development and Stem Cell Biology

Embryonic development is one of the fascinating biological processes in which signaling events are regulated along the spatiotemporal dimension.

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Although much is known about the signaling components involved in developmental processes, the control of these signals via lipid rafts is relatively unexplored. However, in a few studies, the presence of raft-enriched gangliosideGM1 was demonstrated in both preimplantation mouse embryos and mouse oocytes (Comiskey and Warner 2007). Depleting cholesterol in these cells hampered raft formation (Comiskey and Warner 2007; Buschiazzo et al. 2013). Also suppressed is raft-resident c-Src, which is involved in the formation of the second polar body (Buschiazzo et al. 2013). Lipid rafts are also implicated in maintaining the identity and self-renewal of some stem cells. The existence of lipid rafts in mesenchymal stem cells and even embryonic stem cells is extensively recognized, although their regulatory roles remain largely unknown (Lee et al. 2010; Sohn et al. 2018). However, the regulation of hematopoietic stem cells (HSCs) by rafts is better characterized. It was shown that raft-resident integrin, CXCR, and CD117 receptors plays essential roles in the HSCs recognition of their stem cell niche (Alomari et al. 2019; Ratajczak and Adamiak 2015; Wysoczynski et al. 2005).

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Lipid Rafts in Cancer and Metastasis

Cancer is uncontrollable, aberrant cellular proliferation. During carcinogenesis, mutated unhealthy cells are unable to be eliminated by apoptosis. Given the importance of lipid rafts in mitogenic signaling via RTKs and GPCRs, it is not surprising that lipid raft composition is greatly deregulated in tumor cells (Michel and Bakovic 2007; Staubach and Hanisch 2011). Of note, lipid rafts are highly abundant in tumor cells, and lipid rafts existent in tumor cells are highly enriched in cholesterol compared to their untransformed counterparts (LevinGromiko et al. 2014; Li et al. 2006; Owen et al. 2012). Furthermore, it has been established that raft integrity is crucial for proliferative signaling in cancer cells. For example, EGFR recruitment to the rafts was shown to be crucial for proliferation in prostate cancer (Zhuang et al. 2002; Zidovetzki

and Levitan 2007), and complementarily, the disruption of lipid rafts by statins abolishes the EGF signal, leading to apoptosis (Hea et al. 2007; Hryniewicz-Jankowska et al. 2019; Zhuang et al. 2005). IGF signaling and its downstream PI3K/ AKT pathway were affected by lipid raft-mediated regulation. As previously mentioned, AKT activity is controlled through lipid raft-mediated compartmentalization of its activator (PDK1) and negative regulator (PTEN) (Gao et al. 2011). However, this balance is lost in some cancers. For example, in prostate cancer LNCAP cells, raft-resident AKT and non-raft-resident AKT demonstrate different affinities to their substrates (Adam et al. 2007). These AKT pools highlight the presence of two distinct platforms for AKT signaling in the same cell population (Adam et al. 2007). Another example where alterations in raft-mediated regulation of AKT drive tumorigenesis is mantle cell lymphoma (MCL) (Reis-Sobreiro et al. 2013). In MCL cells, AKT is found to be constitutively active (ReisSobreiro et al. 2013; Rudelius et al. 2006), and raft-resident PDK1 and mTOR signaling components favor constant AKT activation (ReisSobreiro et al. 2013). Conversely, lipid raft disruption by alkyl phospholipid edelfosine displaces all AKT, PDK1, and mTOR from lipid rafts, leading to AKT inactivation and apoptosis (Reis-Sobreiro et al. 2013). Similarly, cholesterol extraction through MβCD prevents AKT activation in many transformed cell lines and promotes apoptosis (Calay et al. 2010; Motoyama et al. 2009). Besides being an essential constituent of PM and lipid rafts, cholesterol is the precursor of steroid hormones such as estrogen and androgen (Chimento et al. 2019). Both estrogen and androgen-mediated signaling is associated with the development of gender-specific cancers like breast cancer due to aberrant estrogen signaling and prostate cancer due to aberrant androgen signaling in males (Pelton et al. 2012; Yager et al. 2006). Amplification of estrogen receptor α (ER α) occurs in 70% of all breast cancers (Lumachi et al. 2013). Furthermore, it was demonstrated that cholesterol is a direct agonist of estrogen receptor (Casaburi et al. 2018; Wei et al. 2016). Also, a similar relationship was observed between prostate cancer and cholesterol

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease

in prostate cancer mouse models. When fed high cholesterol diet, these mice develop more aggressive tumors compared to mice fed a regular diet (Llaverias et al. 2010). Canonically, steroid receptors belong to the nuclear receptor family and reside either in the nucleus or cytosol (Pietras and Márquez-Garbán 2007). However, in HeLa cells, a pool of Erα receptors was demonstrated to localize PM. Targeting these receptors to PM occurs through the palmitoylation modification carried out by palmityl-transferase (Acconcia et al. 2005; la Rosa et al. 2012). This pool of ERα receptors was shown to reside in lipid rafts and activate MAPK/ERK and PI3K/AKT axes (Márquez et al. 2006; Maselli et al. 2015). It is interesting to note that androgen receptors have also been shown to cluster close to lipid rafts where AKT signaling activity has been detected (Freeman et al. 2007; Pelton et al. 2012). In addition, lipid rafts are shown to be essential for the metastatic behavior of tumor cells. CD44 is a transmembrane receptor that is involved in cell adhesion. In the clinic, it is used as a metastatic marker for some cancer types (Senbanjo and Chellaiah 2017). Through a mechanism that has been incompletely elucidated, it is believed to regulate the extracellular matrix and induce epithelial-to-mesenchymal transformation (Murai 2015). Proteolytic cleavage of CD44 is necessary for its activity in ECM (Sugahara et al. 2003). CD44 is a raft-resident protein typically, while its protease- ADAM10 resides in non-raft regions (Murai et al. 2011). However, in metastatic tissues, CD44 has been reported to migrate into non-raft areas where it is shed by ADAM10, causing migratory behavior and metastasis (Babina et al. 2014; Murai 2015). The association of lipid rafts/cholesterol with various cancers is an active area of research. The use of drugs that inhibit cholesterol biosynthesis, like statins (Longo et al. 2020), or the use of raftdispersing alkyl phospholipids-like edelfosine (Gajate and Mollinedo 2007; Saraiva et al. 2021; Selivanov et al. 2010; Udayakumar et al. 2016) in cancer treatment is currently being explored and discussed among cancer researchers and clinicians.

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Caveolae

Caveolae is another compartmentalized plasma membrane microdomain described as cholesterol/ sphingolipid-rich lipid rafts with cave-like invaginated morphology (Yamada 1955). The presence of caveolin proteins first molecularly characterized caveolae (Kurzchalia et al. 1992; Rothberg et al. 1992; Way and Parton 1995; Zurzolo et al. 1994). More recently, cavin proteins were discovered to be another significant constituent (Hill et al. 2008). For signal transduction, these specialized cellular morphologies present extensive importance. Several potent receptors were shown to be restricted and concentrated in caveolae, making caveolae a vital signaling hub for the cell (García-Cardeñ et al. 1996; Li et al. 1996a; Liu et al. 1996, 1997; Oh and Schnitzer 2001; Rizzo et al. 1998). The morphological description of caveolae was first made in the gall bladder of mouse epithelium by Yamada and dates to 1955 (Yamada 1955). Later, caveolae were shown to be lipidrich plasma membrane microdomains similar to lipid rafts, but consequent molecular characterization experiments showed that both have distinct molecular components (Liu et al. 1997; Oh and Schnitzer 2001; Zurzolo et al. 1994). The formation of functional caveolae requires the enrichment and oligomerization of a cholesterolbinding protein named Caveolin 1 (Cav1) (Fra et al. 1995; Kurzchalia et al. 1992; Monier et al. 1995; Murata et al. 1995; Rothberg et al. 1992). Cav1 was the first caveolin protein described, followed by the discovery of other homologs, Caveolin 2 (Cav2) and muscle-specific Caveolin 3 (Cav3) (Scherer et al. 1996; Way and Parton 1995). Much later than the discovery of Cav1, another essential caveolar protein-Cavin 1, was discovered (Hill et al. 2008; Liu et al. 2008). Further, Cavin 1 homologs-Cavin 2, Cavin 3, and Cavin 4 were discovered and found to have roles in caveolar function in mammals (Bastiani et al. 2009; Hansen et al. 2009; Kovtun et al. 2014; McMahon et al. 2009). Among all caveolin and cavin proteins, Cav1 and Cavin 1 were shown to be essential for the formation

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of a functional caveolae (Hill et al. 2008; Liu et al. 2008). Caveolae formation starts with binding caveolin proteins to cholesterol in a 1:1 ratio and budding of caveolin-rich vesicles from Golgi network membrane domains (Hayer et al. 2010; Murata et al. 1995). After caveolin accumulation around the cholesterol-rich lipid species, caveolin proteins self-assemble themselves into homo- and hetero-oligomers (Monier et al. 1995). Oligomerized caveolins recruit important lipid species such as phosphatidylserine (PS), phosphatidylinositol phosphate PIP2, sphingomyelin, and gangliosides (Sohn et al. 2018). Caveolin-rich lipid rafts are further tightened and fixated by the recruitment and the binding of Cavin proteins. By following those steps, caveolae reach their well-known striated disc-like appearance under the electron microscope (Monier et al. 1995; Parton et al. 2021; Rothberg et al. 1992).

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Compartmentalized Signaling Through Caveolae

Caveolae is a critical regulatory plasma membrane compartment for signal transduction. The regulation of cellular signaling gets affected by caveolae at two levels. First, preassembled signaling complexes are compartmentalized and enriched in the caveolar membrane. Some of the most studied signaling cascades get initiated from this membrane microdomain, and their receptors are restricted at the caveolar membrane (Martinez-Outschoorn et al. 2015; Okamoto et al. 1998). Insulin, EGF, PDGF, GPCR, and eNOS are some of the well-studied cellular signals that are relayed into the cells through caveolae (Couet et al. 1997b; Liu et al. 1996, 1997; Nystrom et al. 2013; Oh and Schnitzer 2001). Second, the activity of these signaling complexes is regulated by caveolae or, more specifically, caveolar protein Cav1. Over the years of research, it was shown that the receptors of the enlisted signaling pathways above, as well as H-Ras, K-Ras, Src-tyrosine kinase, and heme oxygenase, precipitate together with Cav1 in

co-immunoprecipitation experiments (Li et al. 1996b; Song et al. 1996; Taira et al. 2011). Further molecular characterization of the interaction of Cav1 with the members of these signaling pathways revealed the Caveolin scaffolding domain (CSD), which is mapped to 82–101 residues of Cav1 and regulates all the Cav1associated signaling. CSD was shown to have a negative regulatory role on Cav1-associated signaling. For this inhibitory regulation to occur, CSD binds to the caveolin binding motif (CBM), which is present in many Cav1associated signaling components (Bernatchez et al. 2005; Kirkham et al. 2008; Nystrom et al. 2013; Song et al. 1996; Taira et al. 2011). Although Cav1 interaction was shown to inhibit the basal activation signaling elements such as Src and K-ras (Couet et al. 1997a; Li et al. 1996b), the inhibitory activity of CSD is best characterized in eNOS signaling. First, eNOS hydrophobic pocket, therefore catalytic activity, was shown to be blocked and inhibited by the side-chain extension of phenylalanine 92 (F92) of CSD (Bernatchez et al. 2005; Trane et al. 2014). Moreover, the presence of a peptide identical to CSD was shown to inhibit the eNOS signal. The mutated version of the CSD peptide, which lacks all the aromatic residues (including F92), was found to activate the eNOS signal due to a probable competition between the mutated peptide and Cav1 (Bernatchez et al. 2011). Although CSD is largely acknowledged as the caveolae’s signal regulatory component, there is still some dispute in the caveolae field. The main cause of this contention is CSD itself, which may be buried inside the plasma membrane (Kirkham et al. 2008). Recent studies indicated that Cav1 has a more dynamic structure than initially contemplated and that CSD can exist in multiple states, which might support signaling switches in different contexts (Liu et al. 2016; Sinha et al. 2011). Caveolar proteins other than Cav1 were also found to affect several cellular signals. For example, Cav2 fatty acylation and phosphorylation have a role in regulating insulin signaling pathway. These two post-translational modifications

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease

inhibit the insulin receptor-SOCS3 phosphatase interaction, leading to IRS-1 activation and activated Stat3 translocation to the nucleus (Kwon et al. 2009, 2015; Kwon and Pak 2010). Other studies indicated that Cav3 might function in carrying the membranal signal to the nucleus. For example, C-terminal 154–156 residues of Cav3 were shown to regulate insulin-induced phospho-ERK translocation to the nucleus (Kwon et al. 2011). Interestingly, Cav3 was also found to be crucial for activating the estrogen receptor by the hormone 17β-estradiol (Totta et al. 2016). Cavins as well were found to affect cellular signaling. Firstly, the essential Cavin component of caveolae-Cavin 1 determines the fully functional caveolae pool and, as a result, dictates the location of activated receptors signaling through caveolae (Li et al. 2014; Moon et al. 2014a). Also, Cavin-3 was found to be necessary for ERK activation over AKT activation by keeping caveolae anchored to the cortical cytoskeletal elements through myosin-1c (Hernandez et al. 2013). Caveolae can also affect cellular signaling by regulating the actin cytoskeleton. As a result, it plays a role in lipid sorting and delivery (Echarri and del Pozo 2015). Consequently, caveolae can function in the organization of nanoscale membrane domains, which ultimately control the activation of signaling receptors (Blouin et al. 2016). Mechanical forces can as well affect caveolae dynamics, contributing to the organization of the plasma membrane by the caveolae (Nassoy and Lamaze 2012; Sinha et al. 2011). As an example, c-Src gets activated when caveolar disassembly occurs due to the distribution of Cav1 and membrane lipids like sphingolipids due to membranal stretch (Gervásio et al. 2011). More interestingly, the absence of Cav1 leads to the disorganization of Ras protein, lipid, and phosphatidylserine distribution (Ariotti et al. 2014). Finally, caveolar disassembly was shown to affect changes in Gaq-Cav1 interaction and result in reduced Ca+2 signaling, concomitant with perturbations in the localization of calcium pumps (Fujimoto 1993; Gervásio et al. 2011; Guo et al. 2015).

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Caveolae in Cancer

Caveolar composition and signaling have long been related to cancer. First, it was observed that Cav1 levels decreased and caveolae got lost during the cellular transformation of NIH 3T3 cells after the expression of oncogenes (Koleske et al. 1995). Cav1 expression levels are inversely correlated with the colony size of the transformed cells (Koleske et al. 1995). Also, Cav1 knock-out mice exhibit hyperproliferation in the lung and vascular tissues (Drab et al. 2001; Razani et al. 2001). Next, a role for Cav1 in breast cancer is described. Cav1 deletion in MMTV-PyMT model of breast cancer in mice leads to a delayed initiation of tumor growth, an increase in tumor burden, ERK1 hyperactivation, and cyclinD1 overexpression (Williams et al. 2004). However, cancer progression and survival studies revealed that the main predictor of survival is the Cav1 level in stromal cells rather than epithelia (Sloan et al. 2009; Witkiewicz et al. 2009). The lower the expression of Cav1 in stromal cells, the faster the cancer progresses (Sloan et al. 2009; Witkiewicz et al. 2009). Lisanti and colleagues isolated breast stromal cells (cancer-associating fibroblasts (CAFs)) from patients and compared their metabolism to normal fibroblasts of the matched patient (Martinez-Outschoorn et al. 2015; Pavlides et al. 2009). CAFs represent significant metabolic/ expressional differences compared to normal fibroblasts. Then, they devise a physiological mechanism named the “Reverse Warburg Effect” in which transformed cells induce a differentiation program in stromal cells (MartinezOutschoorn et al. 2015; Pavlides et al. 2009) Therefore, the idea is that cancer cells induce tumor stromal cells to undergo a myofibroblast differentiation. Differentiated tumor stroma activates TGFβ signaling and goes through a metabolic transformation yielding a Warburg metabolism in stromal cells, although present in normoxic conditions. Metabolically altered stroma then sustains the tumor growth and progression by representing metabolites like lactate or pyruvate (Pavlides et al. 2009). Besides Cav1,

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caveolar protein cavin1 is considered a prognostic marker for prostate cancer (Moon et al. 2014b), while caveolar CD36 protein level is associated with breast cancer (DeFilippis et al. 2012).

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Concluding Remarks

After its initial postulation in 1997 (Simons and Ikonen 1997), lipid rafts became a generally accepted concept in recent years. Advances in microscopy techniques revealed that PM is not only “not homogenous” but much more heterogenous than we thought before. There are probably different subtypes of lipid rafts. For example, a two-color PALM study has shown that TCR, Zap70, and LAT proteins are destined to different membrane microdomains upon activating TCR signaling (Sherman et al. 2011). The concept of distinct lipid raft types brings some provoking questions as “How can we differentially mark these raft species molecularly?”, “How proteomics of distinct raft types differ?”, “How can specific proteins are targeted to different raft species?”, “Are these different subcompartments preassembled or co-assembled?” Another outstanding question is, “What would we encounter if we increase lipid raft resolution into atomic levels?”. In other words, “Can we crystalize these solid-like microdomains and determine their structure by X-ray crystallography?”. The crystallization of lipid rafts has been speculated earlier (de Almeida and Joly 2014). Moreover, there were few attempts to crystalize lipid rafts in model membranes, but the data acquired in these studies were restricted (de Almeida and Joly 2014; Park et al. 2020; Ziblat et al. 2012). However, recently developed co-Mesh X-ray crystallography could be a promising method to unravel the structures of lipid rafts. With this technique, serial X-ray images of multiple crystals are acquired and assembled so that they can present information on the dynamic nature of the molecules within the crystals (Sierra et al. 2015). As a result, this approach might be useful for determining the structure of lipid rafts. Now, it is clear that planar lipid rafts and caveolae are both cholesterol-sphingolipid-rich

membrane domains, one favoring the presence of flotillin and the other caveolin. However, we are unaware of how they are specifically localized to the PM, meaning if planar/non-planar rafts favor particular dimensional positioning within the cell. In those terms, we need more precise knowledge of the interactions between rafts and cytoskeleton. Another interesting and not thoroughly answered question is, “Within the same organism, why do some cells acquire caveolae and why do others not?” It is now known that the presence of both Cav 1 and Cavin 1 is a prerequisite for caveolae formation (Parton et al. 2021). However, we are still far from understanding how both proteins integrate at the membrane and enable the typical membrane curvature of caveolae. In some disease conditions, it is now well characterized that lipid raft-mediated signaling gets perturbed. Mainly in many cancer types, it was observed that raft-mediated signaling is deregulated. It can be reflected by the increased cholesterol levels within the whole cells and cholesterol within the rafts of cancers compared to their untransformed counterparts. It is yet unclear what kind of dysregulations take place in the rafts of those cancer cells. For example, we know that when cells are depleted from cholesterol by MβCD, EGFR moves into non-raft regions, and tumorigenic signaling is activated. Nevertheless, quite the opposite effect is observed for other RTKs. For example, raft blockage results in the inhibition of proliferative signaling of IGFR. So, “What specific raft-mediated mechanisms distinguish these akin signaling molecules?” Another topic open to discussion is the disruption of lipid rafts as a therapy against cancer. First, the use of raft inhibiting—alkyl phospholipid-like edelfosine—is being investigated as an anticancer drug (Mollinedo 2014). The anticancer effects of edelfosine are demonstrated in many studies in the cell culture or xenografted animals of several types of tumors (Mollinedo 2014). Also, another edelfosine analog, perifosine, is under investigation for its anticancer effects on hematological cancers and solid tumors (Richardson et al. 2012). A more dramatic approach is using cholesterol synthesis inhibitors-statins for cancer

Rafting on the Plasma Membrane: Lipid Rafts in Signaling and Disease

treatment. Like perifosine, statins are also investigated in numerous clinical trials (di Bello et al. 2020). Given the critical functions of lipid rafts and cholesterol, the value of inhibiting them will become evident in the coming decade.

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Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 109–118 https://doi.org/10.1007/5584_2022_757 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 Published online: 27 December 2022

Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy Giulia Silvani, Valentin Romanov, and Boris Martinac

Abstract

The acoustic force spectroscopy (AFS) tool was recently introduced as a novel tool for probing mechanical properties of biomolecules, expanding the application of sound waves to high-throughput quantification of the mechanical properties of single cells. By using controlled acoustic forces in the piconewton to nanonewton range, tens to hundreds of cells functionalized by attached microspheres can simultaneously be stretched and tracked in real-time with sub millisecond time response. Since its first application, several studies have demonstrated the potential and versatility of the AFS for high-throughput measurements of force-induced molecular mechanisms, revealing insight into cellular biomechanics and mechanobiology at the molecular level. In this chapter, we describe the operation Giulia Silvani and Valentin Romanov contributed equally to this work. G. Silvani School of Material Science and Engineering, University of New South Wales, Sydney, NSW, Australia V. Romanov Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, NSW, Australia B. Martinac (✉) Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, NSW, Australia St Vincent’s Clinical School, University of New South Wales, Sydney, NSW, Australia e-mail: [email protected]

of the AFS starting with the underlying physical principles, followed by a run-down of experimental considerations, and finally leading to applications in molecular and cellular biology. Keywords

Acoustic force spectroscopy (AFS) · Acoustic radiation force · Acoustic standing waves · Adhesion kinetics · Adhesion strength · Biomechanics · Lab-on-a-chip · Mechanobiology · Microfluidic · Single biomolecule · Viscoelasticity

Abbreviations AFS ECM HEK IPSCs LUT NA PDMS RGD

1

Acoustic force spectroscopy Extracellular matrix Human embryonic kidney Induced pluripotent stem cells Lookup table Numerical aperture Polydimethylsiloxane Tripeptide Arg-Gly-Asp

Introduction

Acoustic wave generating instruments are non-invasive, versatile, and cost-effective tools offering excellent potential applications in biological sciences, such as medical diagnostic 109

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imaging (Sarvazyan et al. 2013), drug delivery (Mitragotri 2005), and cell manipulation (Mulvana et al. 2013). Recently, it has been shown that acoustic waves can also be used to exert forces on single molecules, expanding their application in biomechanics and mechanobiology studies. Biological processes are known to be regulated by the behaviour of cells in reaction to the external mechanical environment. Cells are the structural and functional units of all living organisms, which have characteristic mechanical properties (e.g. viscosity and elasticity) that determine the ability of cells to withstand and respond to mechanical forces from the surrounding environment. How cells respond to these external cues will influence their behaviour, generating a complex cascade of cellular signalling events characterized by conversion of mechanical stimuli into intracellular biochemical signals (Eyckmans et al. 2011; Huang et al. 2004). This process of mechanosensory transduction involves a hierarchy of molecular complexes, including the extracellular matrix, membrane lipid bilayer containing force-sensitive molecules, and cytoskeletal network. Understanding the role mechanical stimuli, such as shear force or stretch, play in cellular homeostasis has required the development of new generation of biophysical tools that allow for probing cellular and subcellular mechanical properties at high spatial and temporal resolution on a biologically relevant force scale. However, conventional approaches for evaluating molecular mechanics use to ‘average’ the response of a group of molecules, which results in loosing key details due to the inhomogeneity and stochasticity of the measured ensemble properties. The development of singlemolecule manipulation techniques, such as optical tweezers, magnetic tweezers, and atomic force spectroscopy (Neuman and Nagy 2008), has significantly increased the ability to study mechanical properties at the single molecule level, allowing for detailed investigations of force-induced molecular mechanisms in many biological processes. By exerting external mechanical forces, the displacements and the local forces associated with biological molecules

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are measured with nanometre resolution, providing unprecedented amount of information about the molecular events underlying mechanotransduction processes (Sen and Kumar 2010). Recently, a novel single-molecule force spectroscopy tool, the acoustic force spectroscopy (AFS), has been shown to have multiple advantages over traditional measuring techniques (Kamsma et al. 2016). Importantly, acoustic standing waves generated in microfluidic channels are used to trap a large number of functionalized particles at the acoustic nodes which in turn simultaneously stretch individual tethered biomolecules. The non-invasive nature of acoustic waves and the high-throughput functionality are the two core strengths of the AFS system. Another key aspect of the AFS system is the range of the applied acoustic forces between sub-piconewtons and nanonewtons, which enables researchers to induce conformational changes in biomolecules and monitor related events. It has been shown that the AFS can be used to investigate the strength of DNA-protein interactions (Sitters et al. 2015) as well as the real-time assembly of a virus particle around a DNA (Van Rosmalen et al. 2020). More recently, the AFS was shown to have great potential in measuring forces at cellular level by probing viscoelastic properties of erythrocytes and human embryonic kidney (HEK) cells in response to application of different drugs (Sorkin et al. 2018; Romanov et al. 2021) or the stiffness modulation of endothelial cells subjected to physiological fluid shear stress (Silvani et al. 2021). In the following section, we describe principles of the AFS operation and provide details of critical experimental parameters for applications in molecular and cellular mechanics.

2

Principles of the AFS Operation

Integrated in a lab-on-a-chip device, the AFS system consists of a piezoelectric element excited by an oscillatory voltage which generates a standing acoustic wave within a microfluidic channel made of glass (Fig. 1).

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Fig. 1 General operational principle of the AFS system. A piezoelectric element is used to generate standing acoustic waves within a microfluidic channel of the experimental chip

Any particles (with volume Vp) floating into the channel having different density than the surrounding medium will experience an acoustic pulling force along the vertical (z) direction, which forces them to align at the acoustic node of the standing wave (Fig. 2a). The acoustic radiation force, Frad, applied on each particle is described by the following equation:

at this dependence is to take a force balance around a bead experiencing acoustic radiation within a fluid: F rad: þ F buoyancy - F gravity - F drag = 0 F rad = vp γ brenner -

4 π g r 3 ρp - ρf 3

ð3Þ ð4Þ

with F rad ðzÞ = - ∇U rad ðzÞ

ð1Þ

where Urad is the energy of the acoustic wave defined as: U rad = Vp

1 - k 3 ρ - 1 κf jPðzÞj2 ρ jvðzÞj2 4 4 2ρ þ 1 f ð2Þ

in which P(z) is the acoustic pressure field, v(z) is ρ k the velocity field, and ρ (=ρp) and k (=kpf ) are the f

density ratio and compressibility ratio between the particle and the fluid, respectively (Settnes and Bruus 2012; Gor’kov 1962). The magnitude of the force applied to the microsphere can vary from the sub-piconewton to nanonewton and is determined by several factors, including the material and size of the microsphere, the medium inside the flow cell, the intensity of the acoustic wave, and the vibration of the piezoelectric element (Kamsma et al. 2018). Another way to look

γ brenner =

1-

9r 8h

þ

r3 2h3

6πμr ð5Þ 57r4 r5 7r11 - 100r þ 200h 4 þ 11 5h5

where ρp and ρf are the density of particle and the fluid, respectively, μ is the viscosity of the media where particle is floating with a velocity vp, r is the radius of the particle, and h is its height. The forces acting on a suspended particle in a fluid are the gravity force, the buoyancy force, the Stokes drag, and the acoustic radiation force (Fig. 2b). For a particle moving perpendicularly from the surface of the substrate at constant velocity, these forces cancel out, and the particle will experience an acoustic radiation force directly proportional to the drag force. Indeed, gravity and buoyancy forces are constant, while the drag force is directly related to the particle’s velocity which in turn depends on the amplitude of the standing wave. Large amplitudes result in larger acoustic forces, thus leading to faster bead movement ("vp). Holding the amplitude constant, material properties are the next major determinant of how

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Fig. 2 Particle floating in a fluid upon the application of the acoustic radiation force. (a) Unbound particles will aggregate at nodal points throughout the chip. (b) A particle initially at rest will experience gravitational and

buoyancy forces. Application of an acoustic force will exert radiation force on the particle, causing movement, leading to drag

much force an object will experience. A silica particle, moving through water (ρwater = 1000 kg/m3), with a density of 2000 kg/m3 and a radius of 5 μm experiences a constant gravitational force of ~10 pN and a buoyancy force of ~5 pN. Hence, at least 5 pN of force is required to move the bead from the bottom of the channel. Referring to Eq. (4), the greater the difference between the densities of the object to the surrounding fluid, the greater the acoustic radiation force. Using the example above, ρp - ρf = 2000 - 1000 = 1000. Conversely, a red blood cell with a density of ρrbc = 1110 kg/m3, surrounded by water, will have a difference of ρp - ρf = 110. Hence, to move a red blood cell with the same velocity as a silica bead, larger acoustic radiation force will need to be applied. The microfluidic channel can be functionalized with several biologically interesting structures such as proteins or cells. Properties of either the protein or the cell can be probed via the attachment and movement of a functionalized bead (Fig. 3a), the latter being used as force transducers when excited by an acoustic wave at the proper frequency. The resonance frequency – the optimal excitation frequency of the system – is in MHz for a microsphere with diameter in the micrometre range. By tracking the microsphere’s displacement using a digital camera and comparing the displacement of the microspheres with the

magnitude of the applied force, hundreds of force/ deformation curves are simultaneously collected using the AFS system and correlated with molecular structures, molecular bond strength, or the mechanical properties of a cell membrane. The slow deformation of a viscoelastic structure (Fig. 3b), such as a biological cell, is described by the creep function, which captures cell deformation as a function of constant force over a predefined time period. First described in 2015 (Sitters et al. 2015), the AFS has been greatly improved, resulting in better optical and acoustic performance (Kamsma et al. 2016). First, a newly developed transparency piezo element improved the microsphere tracking accuracy as well as the measurable field of view, due to the compatibility of the AFS with trans illumination imaging. It has been shown that AFS can also be integrated with epifluorescence microscopes, in combination with high numerical aperture (NA) water and oil-immersion objectives, expanding its measurements applicability. However, this was only possible with a steep reduction in force magnitude (Kamsma et al. 2016). Second, the acoustic properties of AFS were improved by optimizing the thickness of the channel depth in order to generate a more efficient force at the bottom of the microfluidic channel, where cells and molecules are seeded. Finally, an external pump allows for complete

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Fig. 3 Application of acoustic force spectroscopy in single-molecule and single-cell force measurements

control over fluid flow rate and pulsating conditions, better mimicking in vivo conditions (Silvani et al. 2021). Among the advantages, the AFS has several limitations. The optical setup determines the field-of-view, limiting the number of microspheres that can be tracked in real-time. Further, variations in bead size will affect the acoustic radiation force magnitude across the field-of-view. Also, prolonged measurements, at high operating voltages, can raise temperature, which may lead to sample heating. Nevertheless, at a maximum voltage of 42 V at 13 MHz applied for less than a few seconds, the resultant sample heating is less than 2 °C, a rise in temperature acceptable for most measurements of mechanical properties of cells, for example (Kamsma et al. 2018). To better understand the functionality and applicability of AFS as a tool, the following section focuses on the experimental setup, providing details about critical AFS parameters, microsphere functionalization and tracking, and the model used to correlate deformational information to viscoelastic cell properties.

3

Experimental Setup and Analysis

3.1

Resonance Frequency

The resonance frequency is the optimal excitation frequency of the system. The resonance frequency depends on several factors, including channel geometry, temperature, and fluid properties. In the case of the AFS system, a

small change in the thickness of the fluid layer will have minimal impact on the resonance frequency but can influence the resultant force output (Kamsma et al. 2016). Because the thickness of the microfluidic channel is constant, the main variable to consider is the temperature of the system. The AFS chip (Fig. 1) can be heated from room temperature up to about 40 °C. Each degree change in temperature changes the resonance frequency of the system. The resonance frequency of an AFS chip can be determined by measuring the point of maximum displacement of a bead, measured from the surface of the channel. Each AFS chip comes pre-calibrated from the factory (water at 21 °C), using this frequency as the starting point, the actual resonance frequency is adjusted in steps of 0.01 MHz, either up or down (Fig. 4a). The change in resonance frequency as a function of increasing temperature is shown in Fig. 4b.

3.2

Temperature

Biological cells exhibit temperature-dependent properties, that is, temperature may drive changes in the overall elasticity and/or fluidity of the cell. These two properties are a measure of the viscoelasticity of the cell, the properties of which are also dependent on the cell area where the viscoelasticity is being measured. For example, viscoelasticity at the cell periphery may be predominantly driven by actomyosin cytoskeletal arrangement and may differ from the properties measured closer to the nucleus, where stresses are born not only by the nucleus but also by the cytoskeleton

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Fig. 4 Determining resonance frequency. (a) Maximum bead displacement as a function of frequency. The resonance frequency is the point at which the z-extension is

maximum. Temperature is held constant. (b) Frequency vs temperature plot

(Sunyer et al. 2009). The absolute values of elasticity and fluidity are difficult to characterize as they are cell type dependent, influenced by passage number, cell cycle, media composition, etc. Several studies have been carried out to probe the effects of temperature on the viscoelasticity of several different cell lines with some studies showing an increase in stiffness and a decrease in fluidity, while other studies showing the opposite trend, a decrease in stiffness and an increase in fluidity (Romanov et al. 2021; Sunyer et al. 2009; Sunnerberg et al. 2019; Chan et al. 2014). One potential issue with an acoustic driver is localized heating arising from the conversion of the mechanical energy into heat. Particularly relevant to the AFS measurements is the presence of a heatsink between the piezoelectric elementglue-glass and liquid layer within the microfluidic channel (Wiklund 2012). If thermal effects do arise, they tend to be present close to the surface of the glass channel, typically occupied by the object of study (DNA, cells, etc.). The greater the amplitude, the greater the chance of localized heating effects that may alter cell viscoelasticity (Nguyen et al. 2021), introducing a complex source of error. No systematic study utilizing the AFS system has been carried out to investigate the influence of increased amplitude, and hence temperature, on cell viscoelasticity.

3.3

Microsphere Coating

Depending on the experimental requirements, beads can be coated with a variety of molecules to promote adhesion to cells. Typically, fibronectin at a final concentration of 100 μg/mL is used for coating. Fibronectin can be used to coat both the glass microfluidic chip and the silica beads. The type and concentration of the coating is also dependent on the cell type. Different molecules can be used to study different aspects of the system. The physical properties (material and volume) of the beads play an important role in how the cell interacts with the bead. Smaller beads (6 μm) (Grinnell and Geiger 1986). While larger beads may not be phagocytosed to the same degree, partial ingestion or localized accumulation of actin patches have been demonstrated (Grinnell and Geiger 1986; Deng et al. 2005). The bead coating plays an important role in the cell structure that is ultimately probed by AFS or any method used for studies of molecular and cellular mechanics. For example, magnetic beads coated with RGD (peptide with sequence Arg-Gly-Asp) had the greatest effect on cytoskeletal rearrangement while also measuring stiffness values tenfold greater than the same beads coated with

Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy

acetylated low-density lipoprotein (Puig-deMorales et al. 2004). Further consideration needs to be given to the coating of the microfluidic channel. We have found that fibronectin is sufficient for use with HEKT293T and endothelial cells (Romanov et al. 2021; Silvani et al. 2021). However, other extracellular matrix (ECM) components, such as collagen, laminin, and gelatin may sometimes be required to achieve the optimal attachment. Among those mentioned here, fibronectin and laminin are two of the most commonly employed ECM molecules that have been shown to greatly enhance attachment and proliferation of humaninduced pluripotent stem cells (IPSCs) on polydimethylsiloxane (PDMS) surfaces, as compared to either collagen or gelatin (Yoshimitsu et al. 2014). Typically, these molecules are delivered to the surface of the substrate via diffusion and by chemical modification of the surface to promote binding. There are several limitations to these approaches including lack of control over patterning (non-uniformity), protein denaturing and mis-orientation, as well as protein density variations across the substrate (Cooke et al. 2008).

3.4

Microsphere Tracking

The AFS software utilizes a lookup table (LUT) for tracking the vertical displacement of particles within the microfluidic channel. The LUT is used to generate a z-stack of images, capturing the radial profile of the bead at pre-defined intervals. For typical cell work, 100 nm steps are used with a maximum height of 10 μm. The tracking accuracy will vary depending on experimental conditions (beads, cell type, buffer, etc.). For example, the z-positional resolution for microspheres in a simple buffer has been shown to be around 4 nm (Kamsma et al. 2016). However, in the presence of other objects, such as red blood cells, the resolution has been shown to reduce to about 20 nm (Sorkin et al. 2018). One of the main challenges with using the LUT is tracking of the bead over large distances. The tracking algorithm may clearly identify the bead

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in the first half of the LUT formation and then skip to a nearby object. As such, care should be taken to select the appropriate focus point on the bead, which typically is not the centre of the bead (Sarvazyan et al. 2013).

3.5

Determining Viscoelasticity

A variety of different models can be utilized to measure the creep response of cells. The linear viscoelasticity model utilizes springs to capture elastic responses and dashpots to capture viscous components. While simple and intuitive to use, the number of free-fit parameters makes it hard to assign a specific or a unique biological response to each parameter. On the other hand, the power law model contains two free-fit parameters, one describing the elasticity and the other describing the fluidity. The nature of the fluidity component is yet to be satisfactorily described, but a number of models have been proposed (Kollmannsberger et al. 2011a). In AFS experiments, we use the power law model to capture the creep response of cells under constant force loading conditions. In this experiment, the AFS is used to drive a bead to the acoustic node. If the bead is attached to the cell, the movement of the bead pulls a plasma membrane tether from the cell. The movement of the bead is tracked in real-time, yielding the strain. The power law model describes the change in material compliance with time (Kollmannsberger et al. 2011a): J ðt Þ = J 0

t τ

β

ð6Þ

where J0 is the material compliance, which is inverse of Young’s modulus at time τ describing the normalizing time (t), usually set to 1 s, and β is the power-law exponent. Note that when β approaches zero, Eq. (6) will describe the deformation of a purely elastic material, which corresponds to the stiffness of the material, whereas when β approaches unity, then the equation describes the deformation of a purely viscous material having properties of a Newtonian fluid (Kollmannsberger et al. 2011b).

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The creep compliance is fit to the above expression using (Kollmannsberger et al. 2011b): J ðt Þ =

zðt Þ  πr F

ð7Þ

where z(t) is the extension curve (z-height), F is the force, and r is radius of the particle. This expression is plotted as a function of time, and the power law model is then fitted to the resultant distribution allowing for the values of J0 and β to be extracted for each cell. In addition to the above model, fractional viscoelastic models for power law materials are also gaining popularity. Fractional models can accurately capture the response of viscoelastic materials across a number of time scales, using less parameters than traditional linear models. While the mathematical formalism is outside the scope of this article, we simply present the fractional model of creep compliance here: J ðt Þ =

1 tβ cβ Γð1 þ βÞ

ð8Þ

where cβ represents the hardness of a material, Γ is the gamma function, t is time, and β is the power law exponent, where if β = 0, the material reduces to a simple elastic spring while for β = 1 the material reduces to a dashpot. For more information, we point the reader to this excellent review (Bonfanti et al. 2020).

4

Applications: Single-Cell and Single-Molecule Force Spectroscopy

Acoustic force spectroscopy is the latest addition to the myriad of other tools available for measuring the viscoelastic properties of single cells. Here, we aim to familiarize the reader with the various specific applications of the AFS system.

4.1

Measuring DNA Binding Activity

The AFS instrument was first used to demonstrate the stochasticity and binding activity of RecA

filament protein to DNA (Sitters et al. 2015). As RecA activity is driven by DNA tension, binding and unbinding events under different loading conditions drive changes in tension, thus resulting in altered DNA extension profiles (Sitters et al. 2015). More recently, Van Rosmalen et al. (2020) utilized the AFS to follow, in real-time, assembly of virus-like particles (VLP) on DNA templates. Assembly of virus-like particles was monitored by comparing the force distribution on a bead tethered to DNA and its extension over time. Incubation of DNA + WT fragments of VLPs resulted in both reduction in the force and extensional distance of DNA.

4.2

Measuring Non-adherent Cells

Red blood cells circulate in the circulatory system throughout the body. The passage of a red blood cell through tight constrictions is driven by its ability to deform and to regain shape while retaining function. Diseases such as malaria have been shown to alter the deformability of red blood cells, making them stiffer. In one of the first demonstrations, the AFS was used to measure the mechanical properties of these circulating cells (Sorkin et al. 2018). The instantaneous elastic elongation (F/k1 where k1 is a spring constant in Burger’s model) of red blood cells was used as a measure of elasticity and a way to differentiate between different pharmacological treatments. The AFS was used to demonstrate quantifiable differences in the elastic properties of red blood cells exposed to 0.04% formaldehyde.

4.3

Measuring Cell Adhesion Strength

The AFS is typically used to drive a bead attached to the object of interest. However, the same principles can be applied to moving the object of interest (if not immobilized on the surface). Kamsma et al. (2018) demonstrated an approach for measuring the binding kinetics and adhesion

Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy

forces of CD4+T lymphocytes to fibronectin. A linear force ramp was employed to measure the binding strength starting from ~10 pN up to ~100 pN. Importantly, it was demonstrated that around 300 cells could be tracked in real-time, simultaneously.

force distribution within a single field of view, potential heating effects, and the overall impact of media properties, frequency, and temperature on the final force distribution.

5 4.4

Measuring Adherent Cells

Recently, several articles have been published looking at the viscoelastic properties of adherent cells. Romanov et al. (2021) explored the creep response of adherent cells by measuring their elasticity and fluidity. The AFS instrument was used to measure the viscoelasticity of HEK293T cells under a variety of conditions and in the process demonstrating the effects of temperature, force, and pharmacological treatments on cell elasticity and fluidity. Significantly, changes in elasticity of cells overexpressing a mechanosensitive ion channel protein, Piezo1, could also be captured by the AFS system. While the above study was performed under static conditions, dynamic perfusion is required for the formation of functional, mature endothelial cell monolayers. Silvani et al. (2021) developed further protocols and methods for characterizing the viscoelastic properties of two different types of endothelial cell lines, human aortic and human umbilical vein endothelial cells. Cells were cultured within the AFS chips for a period of 48 h under a constant flow rate of 6 dyn/cm2. The authors reported immunofluorescent staining protocols and an approach for quantifying force-dependent stiffness of the membrane cortex. Nguyen et al. (2021) expanded the utility of the AFS system by developing a new technique for frequency-dependent measurements of the viscoelasticity properties of human umbilical vein endothelial cells. The complex shear modulus was measured over a period of several hours after exposing cells between 70 and 100 h of fluid flow at 1.66 μL/min. Several shortcomings in the fundamental operation of the AFS instrument were also identified relating to non-homogenous

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Conclusions

The AFS instrument is just one of several techniques that employ sound waves to interact with physical objects. The power behind this approach lies in its ability to generate small (>1 pN) and large (> 1 nN) forces that interact with objects in a high-throughput manner, where hundreds or even thousands of objects can be moved and tracked in real time. As such, the AFS system is an excellent case study for the use of ultrasound waves in biology. Although a recent innovation, this technique has been successfully used to study DNA binding events and to characterize the viscoelasticity of a variety of different cell types exposed to many different physical and chemical conditions. We envision that future studies using AFS instrument and future advancements of the system will allow for deeper insights into the mechanobiology behind biological processes.

References Bonfanti A, Kaplan JL, Charras G, Kabla A (2020) Fractional viscoelastic models for power-law materials. Soft Matter 16(26):6002–6020 Chan CJ, Whyte G, Boyde L, Salbreux G, Guck J (2014) Impact of heating on passive and active biomechanics of suspended cells. Interface Focus 4(2): 20130069 Cooke MJ, Phillips SR, Shah DS, Athey D, Lakey JH, Przyborski SA (2008) Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins. Cytotechnology 56(2):71–79 Deng L, Fairbank NJ, Cole DJ, Fredberg JJ, Maksym GN (2005) Airway smooth muscle tone modulates mechanically induced cytoskeletal stiffening and remodeling. J Appl Physiol 99(2):634–641 Eyckmans J, Boudou T, Yu X, Chen CS (2011) A hitchhiker’s guide to mechanobiology. Dev Cell 21(1):35–47

118 Gor’kov LP (1962) On the forces acting on a small particle in an acoustical field in an ideal fluid. Sov Phys Dokl 6: 773–775 Grinnell F, Geiger B (1986) Interaction of fibronectincoated beads with attached and spread fibroblasts: binding, phagocytosis, and cytoskeletal reorganization. Exp Cell Res 162(2):449–461 Huang H, Kamm RD, Lee RT (2004) Cell mechanics and mechanotransduction: pathways, probes, and physiology. Am J Phys Cell Phys 287(1):C1–C1 Kamsma D, Creyghton R, Sitters G, Wuite GJ, Peterman EJ (2016) Tuning the music: acoustic force spectroscopy (AFS) 2.0. Methods 1(105):26–33 Kamsma D, Bochet P, Oswald F, Alblas N, Goyard S, Wuite GJ, Peterman EJ, Rose T (2018) Single-cell acoustic force spectroscopy: resolving kinetics and strength of T cell adhesion to fibronectin. Cell Rep 24(11):3008–3016 Kollmannsberger P, Mierke CT, Fabry B (2011a) Nonlinear viscoelasticity of adherent cells is controlled by cytoskeletal tension. Soft Matter 7(7):3127–3132 Kollmannsberger P, Mierke CT, Fabry B (2011b) Nonlinear viscoelasticity of adherent cells is controlled by cytoskeletal tension. Soft Matter 7(7):3127–3132 Mitragotri S (2005) Healing sound: the use of ultrasound in drug delivery and other therapeutic applications. Nat Rev Drug Discov 4(3):255–260 Mulvana H, Cochran S, Hill M (2013) Ultrasound assisted particle and cell manipulation on-chip. Adv Drug Deliv Rev 65(11–12):1600–1610 Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5(6):491–505 Nguyen A, Brandt M, Muenker TM, Betz T (2021) Multioscillation microrheology via acoustic force spectroscopy enables frequency-dependent measurements on endothelial cells at high-throughput. Lab Chip 21(10):1929–1947 Puig-de-Morales M, Millet E, Fabry B, Navajas D, Wang N, Butler JP, Fredberg JJ (2004) Cytoskeletal mechanics in adherent human airway smooth muscle cells: probe specificity and scaling of protein-protein dynamics. Am J Phys Cell Phys 287(3):C643–C654 Romanov V, Silvani G, Zhu H, Cox CD, Martinac B (2021) An acoustic platform for single-cell, high-

G. Silvani et al. throughput measurements of the viscoelastic properties of cells. Small 17(3):2005759 Sarvazyan AP, Urban MW, Greenleaf JF (2013) Acoustic waves in medical imaging and diagnostics. Ultrasound Med Biol 39(7):1133–1146 Sen S, Kumar S (2010) Combining mechanical and optical approaches to dissect cellular mechanobiology. J Biomech 43(1):45–54 Settnes M, Bruus H (2012) Forces acting on a small particle in an acoustical field in a viscous fluid. Phys Rev E 85(1):016327 Silvani G, Romanov V, Cox CD, Martinac B (2021) Biomechanical characterization of endothelial cells exposed to shear stress using acoustic force spectroscopy. Front Bioeng Biotechnol 4(9):21 Sitters G, Kamsma D, Thalhammer G, Ritsch-Marte M, Peterman EJ, Wuite GJ (2015) Acoustic force spectroscopy. Nat Methods 12(1):47–50 Sorkin R, Bergamaschi G, Kamsma D, Brand G, Dekel E, Ofir-Birin Y, Rudik A, Gironella M, Ritort F, RegevRudzki N, Roos WH (2018) Probing cellular mechanics with acoustic force spectroscopy. Mol Biol Cell 29(16):2005–2011 Sunnerberg JP, Moore P, Spedden E, Kaplan DL, Staii C (2019) Variations of elastic modulus and cell volume with temperature for cortical neurons. Langmuir 35(33):10965–10976 Sunyer R, Trepat X, Fredberg JJ, Farre R, Navajas D (2009) The temperature dependence of cell mechanics measured by atomic force microscopy. Phys Biol 6(2): 025009 Van Rosmalen MG, Kamsma D, Biebricher AS, Li C, Zlotnick A, Roos WH, Wuite GJ (2020) Revealing in real-time a multistep assembly mechanism for SV40 virus-like particles. Sci Adv 6(16):eaaz1639 Wiklund M (2012) Acoustofluidics 12: biocompatibility and cell viability in microfluidic acoustic resonators. Lab Chip 12(11):2018–2028 Yoshimitsu R, Hattori K, Sugiura S, Kondo Y, Yamada R, Tachikawa S, Satoh T, Kurisaki A, Ohnuma K, Asashima M, Kanamori T (2014) Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions. Biotechnol Bioeng 111(5):937–947

Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 119–130 https://doi.org/10.1007/5584_2023_765 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 Published online: 22 February 2023

Drug Therapeutics Delivery to the Salivary Glands: Intraglandular and Intraductal Injections Akram Abdo Almansoori, Arvind Hariharan, Uyen M. N. Cao, Akshaya Upadhyay, and Simon D. Tran Abstract

Salivary gland hypofunction and xerostomia following pathological conditions like Sjogren’s syndrome or head and neck radiotherapy usually lead to tremendous impairment of oral health, speech, and swallowing. The use of systemic drugs to alleviate the symptoms of these conditions has been associated with various adverse effects. Techniques of local drug delivery to the salivary gland have grown enormously to address this problem properly. The techniques include intraglandular and intraductal injections. In this chapter, we will provide a review of the literature for both techniques while incorporating our lab experience in using them. Keywords

Drug therapeutics · Head and neck radiotherapy · Intraductal · Intraglandular · Salivary gland · Salivary hypofunction · Sjogren’s syndrome · Xerostomia

Abbreviations AdMSCs ALS AMI AMY BMSC BoNT, BTXA FDA H&E ID IG IV MSCs NSSVAC PBS PEG PLGA RI SDS SGs SMG

Adipose tissue derived mesenchymal stem cells Amyotrophic lateral sclerosis Amifostine Alpha-amylase Bone marrow stem cells Botulinum toxin Food and Drug Administration Hematoxylin and eosin Intraductal Intraglandular Intravenous Mesenchymal stem cells Immortalized human salivary gland acinar cells Phosphate-buffered saline Poly(ethylene glycol) Poly(lactic-co-glycolic acid) Rho kinase inhibitor Sodium dodecyl sulfate Salivary glands Submandibular salivary gland

Authors Akram Abdo Almansoori and Arvind Hariharan have equally contributed to this chapter.

1 A. A. Almansoori, A. Hariharan, U. M. N. Cao, A. Upadhyay, and S. D. Tran (✉) McGill Craniofacial Tissue Engineering and Stem Cells Laboratory, Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada e-mail: [email protected]

Introduction

Various disorders can affect the salivary glands (SGs) and they range from infections, obstructions, and tumors to autoimmune diseases (Ogle 2020). 119

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Despite the advances in systemic drug administration, it is still not perfectly reliable in terms of efficiency and associated adverse side effects (Muthumariappan et al. 2019; Diggelmann and Hoffman 2015; Teymoortash et al. 2007; Lynggaard et al. 2022). Recently, several local delivery approaches have been conducted for the treatment of salivary gland diseases. This review addresses these efforts using intraglandular (IG) and intraductal (ID) injections. For each approach, the methodology, targeted salivary cells or glands, the used drug, and the efficiency are fully discussed.

2

Intraglandular (IG) Injection for Drug Delivery

Intraglandular (IG) injections involve the process of injecting medication and various other cell therapies into the SGs. The advantage of this injection technique is that it allows for direct access to the SGs as well as reduction of the systemic side effects that are associated with many therapies. It is also less time-consuming and requires less of a dosage to be injected. The objectives of this section are: 1. To discover the different methods of IG injections that have been employed so far and the SGs they have targeted. 2. To review the literature on the different drugs and gene therapeutics that are currently being used or under development and their safety and efficiency. In addition to the above objectives, we would like to introduce a novel transcutaneous non-surgical IG injection technique that can be used to deliver drugs to the submandibular SGs and how we intend to use this technique with radioprotective drug, amifostine (AMI).

2.1

Methods

IG injection is usually given surgically or non-surgically. The surgical route is primarily used for the submandibular SGs and involves a

conservative horizontal incision in the neck area to expose and localize the SGs of interest (Tran et al. 2013). The non-surgical route, however, is the more common route that is employed, usually for the treatment of sialorrhea with botulinum toxin (Mueller et al. 2022). Injections are currently most performed using an ultrasoundguided approach to view the site of injection in the SGs (Mueller et al. 2022). Non-ultrasoundguided IG injections have been used earlier but have fallen out of contention due to less accuracy. A study by So et al. compared the accuracy between ultrasound-guided and non-ultrasoundguided IG injections by visual confirmation of dye accumulation in the submandibular and parotid SGs (So et al. 2017). They found that there was a marked reduction in the accuracy of the non-ultrasound-guided injections in comparison to the ultrasound-guided approach (So et al. 2017).

2.1.1

IG Non-surgical Transcutaneous Approach Recently, our laboratory has been working on a novel IG technique for the submandibular glands (SMGs) to deliver therapeutic agents to mitigate radiation injury to the SGs in mice. The rationale behind developing this technique was to investigate a less invasive and localized method of delivering therapeutics directly to the submandibular SGs to mitigate side effects induced by systemic administration. Moreover, ultrasoundguided injections are usually operator dependent and require an experienced radiologist, which is not possible in many cases. Our laboratory, therefore, looks to develop an anatomical landmarkguided IG technique. To localize the right and left submandibular SGs, the mice were anaesthetized with a ketamine cocktail, and the hair was shaved in the neck region from the chest wall to the lower border of the mandible. A midline was drawn from the lower lip border passing through the inferior border of the mandible up to the chest wall. An incision was made to expose the right and left SMG, and the distance between the midline and the center of the gland was measured to determine the injection site. The mice were sacrificed, the glands were then harvested, and

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the thickness was measured using an endodontic file with a rubber stopper to mark on the syringe to be used for the injection (Fig. 1). According to our preliminary results, the mean total distance of the midline from the lower lip to the chest wall was 1.9 cm, and on exposure of the SGs, the center of the gland was located 0.9 cm from the chest wall and 0.2 cm lateral to the midline. This meant that the center of the SGs was located at half the distance of the midline. As the gland thickness was 0.2 cm, and the thickness of the mouse skin was 0.1 cm, we concluded that the injection depth is 0.3 cm. Using the measurements

above, the IG injection was verified by injecting Trypan Blue dye at a 90° angle to ensure that the drug reached the maximum areas of the gland. It was found that the Trypan Blue solution entered the gland at a success rate of 80%. While the success rate is encouraging, we do anticipate that there will be limitations. Due to the health or size of the mice, there could be variations in the SG location measurements, which could affect the area of administration. It also remains to be seen if there will be any difference if substances are injected in the center versus other areas of the gland, thus necessitating further studies.

Fig. 1 Demonstration of IG injection using a mouse animal model. (a) The mouse’s neck was shaved and outlined. (b) The distance from the base of the mandible to the sternum was measured. (c) The submandibular salivary glands were exposed and the distance from the neck midline

to the center of the salivary gland was measured. (d) The thickness of the salivary gland was measured. (e) A mouse with both SMGs that were exposed after transcutaneous injection. (f) The glands were harvested to examine their saturation with the injected blue dye

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2.2

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Targeted Glands

IG injections have traditionally been employed in the parotid and SMGs; however, there are no reports of the involvement of the sublingual SG or minor SGs. Most studies that involve the injection into the parotid use non-ultrasound IG injections based on superficial landmarks, whereas ultrasound-guided injection is used for the SMGs (So et al. 2017). The injection site for the parotid is usually marked behind the ascending ramus or before the mastoid process (So et al. 2017; Jost et al. 2019). In the SMG, a reference line is made from the gnathion to the mandibular angle, following which, the injection site is marked at a distance that is 20–35% from the angle, 1.5 cm below the inferior mandible (So et al. 2017). In the ultrasound-guided approach for the SMG, the IG injection is given from a lateral approach, where the head of the patient is facing away from the injection site and the needle is inserted perpendicular to the ultrasound transducer (So et al. 2017).

2.3

Drugs and Agents Used

1. Botulinum Toxin Botulinum toxin A (BoNT) is an FDA-approved drug that is indicated for aesthetic as well as clinical applications (Sherif et al. 2018). It is a neurotoxin that is produced by Clostridium botulinum and was first used for the treatment of strabismus and then subsequently, for disorders involving spasticity and cosmetic purposes (Nigam and Nigam 2021; Scott 1981). Bushara et al. in 1997 were the first group of researchers to use it in the treatment of sialorrhea or excessive salivation through surgical IG administration (Bushara 1997). It has, since then, been employed in neurological disorders such as Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and cerebral palsy, all of which have clinical features of sialorrhea (Fuster Torres et al. 2007). Traditionally, BoNT has been injected in either the parotid gland only, the submandibular gland only, or both glands, and it is still unclear as to which of these three scenarios is most effective (Ellies and Laskawi 2010).

In addition to Bushara’s study, BoNT first displayed its effects of alleviating sialorrhea in patients with Parkinson’s disease in a study by Pal et al., with minimal side effects and through intra-parotid injections (Pal et al. 2000). The potential of BoNT was further studied in a metaanalysis where it was found to significantly reduce drooling in both adults and patients with sialorrhea (Vashishta et al. 2013). Further studies confirmed the effectiveness of BoNT in treating sialorrhea by examining the histological changes in the SGs. Teymoortash et al. examined the structural and functional changes of IG administration of BoNT in acinar cells in rats. They found that there was atrophy of the acini, which they concluded was because of glandular denervation, explaining the reduced salivary secretion (Teymoortash et al. 2007). This was confirmed in a more recent study by Sherif et al., who observed that the acini lost their spherical morphology in the group of mice that received BoNT; however, they also noted that the morphology was recovered over time (Sherif et al. 2018). Recent studies have interestingly shown that while BoNT has uses in reducing salivary secretion, it has potential applications in radioprotection. Ultrasound-guided IG administration was able to offer radioprotection for both submandibular and parotid glands with minimal side effects. A clinical trial showed that injection of BoNT preserved gland function in patients undergoing radiotherapy for head and neck cancer, and further studies should explore the safety and efficacy of IG BoNT (Teymoortash et al. 2016). BoNT is also associated with some disadvantages, such as high costs and the development of antibodies against the botulinum toxin (Lakraj et al. 2013). Studies have shown that although uncommon, BoNT injections could lead to an increase in salivary thickness, dysphagia, pneumonia, and xerostomia (Vashishta et al. 2013; Lakraj et al. 2013). 2. Stem Cell Transplantation A novel area of research includes the IG administration of stem cells to mitigate SG hypofunction, mainly due to radiotherapy. IG administration with stem cells has so far been employed in the submandibular SG through the

Drug Therapeutics Delivery to the Salivary Glands: Intraglandular and Intraductal Injections

surgical approach. Lombaert et al. were one of the pioneers in initiating the importance of stem cell transplantation into the salivary glands, by identifying markers that determined stem cell differentiation into acinar and ductal cells (Lombaert et al. 2008). Building on this concept, they were able to further enrich the cells using a progenitor cell marker, c-kit, to promote long-term restoration of an irradiated submandibular mouse SG in vivo (Lombaert et al. 2008). These findings contributed to future studies using different types of stem cells to be transplanted in the SGs through the IG route (Nanduri et al. 2013; Isabelle et al. 2013). Bone marrow stem cells (BMSCs) and their extracts are an emerging research area for stem cell transplantation. Differentiation of BMSCs into salivary gland acinar cells have been observed, and the school of thought is that this is due to the secretion of paracrine factors like cytokines and growth factors, which are required for tissue regeneration and repair (Fang et al. 2015). While studies have displayed the success of BMSCs when given intravenously (IV), IG administration is still poorly understood (Fang et al. 2015; Tsutsui 2020). One of the first studies to test BMSCs on SGs was by Tran et al., where they showed that IG administration of BMSC from mice was able to restore SG hypofunction when administered post-irradiation (Tran et al. 2013). The IG method was compared to the IV approach and found that the number of injections and dosage to provide the same effects was significantly reduced (Tran et al. 2013). This laid the foundation for future studies on BMSC and more recent studies using mouse BMSC extracts agreed with previous findings, showing that they were able to ameliorate SG hypofunction post-IR at different time points when given IG (Lim et al. 2013; Mohamed et al. 2022). A study by Schwarz et al. also compared the IG administration of BMSC to intravenous (IV) administration in a surgical model of submandibular SG damage in mice and found that the presence of MSCs, leucocytes, and macrophages was more enhanced in the damaged SG when given IG, thus demonstrating the potential of this injection technique (Schwarz et al. 2014). In addition, a study

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by Rabea et al. demonstrated the regenerative capacity of BMSCs when given IG to the parotid glands of rats, which shows that the IG technique using stem cells could be useful for parotid glands as well (Rabea et al. 2022). A very recent study found that BMSCs derived from humans could also prevent SG hypofunction in mice post-IR when given IV, which has created avenues for testing BMSCs derived from humans for IG administration (Su et al. 2018). The IG approach to preventing SG hypofunction has also been tested with stem cells from different sources. Adipose tissue MSCs (AdMSCs) have also been explored for tissue regeneration studies and are known to maintain characteristics of multipotent progenitor cells (Kim et al. 2019). Kim et al. examined whether AdMSCs could restore radiation-induced SG hypofunction by IG injection in mice and found that there were elevated expressions of epithelial markers, suggesting that localized delivery could regenerate SG damage (Kim et al. 2019). This was confirmed by Wang et al., who showed that AdMSCs along with plasmaderived fibrin extracts could restore SG hypofunction when given IG post-IR (Wang et al. 2017). Dental-pulpal stem cells have also been tested for their capacity to regenerate SG defects and are known to be less invasive to isolate than BMSC. A study on rats with induced-diabetic SG defects showed that when dental-pulpal stem cells were given by IG transplantation, they could restore the defects through reduced vacuolization of acinar cells and an increase in serum markers (Narmada et al. 2019). A unique study isolated and cultured stem cells from minor salivary glands (labial glands) in mice and injected them IV and found that they could mitigate injury to the SGs, which opens avenues for testing this method by IG administration (Su et al. 2020). The success of stem cell transplantation has culminated with the start of in-human studies using the IG technique. Sumita et al. is currently performing the first-in-human transplantation of mononuclear cells using ultrasound-guided IG injection to submandibular SGs to mitigate injury due to irradiation (Sumita et al. 2020). It

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is expected to be the first instance of using a less invasive cell-based therapy for SG regeneration. Comella et al. performed the first-in-human clinical trial wherein they delivered AdMSCs with plasma-derived extracts by ultrasound-guided IG injection to both the parotid and submandibular glands and found that it improved the quality of life of patients undergoing radiotherapy as well as an increase in gland size (Comella and Bell 2017). A Danish-based clinical trial (MESRIX) is currently underway to test the effects of allogeneic AdMSCs to treat radiation-induced xerostomia through the IG injection (Lynggaard et al. 2022; Grønhøj et al. 2017). They have so far shown that it can be a feasible method of injection into the parotid and submandibular SGs with few adverse effects months after treatment and there was a significant increase in stimulated and unstimulated salivary flow rates (Lynggaard et al. 2022; Grønhøj et al. 2017). Positive results have shown that stem cell transplantation using the IG technique can be further investigated in larger clinical trials safely and effectively. 3. IG Administration of Amifostine (AMI) to Prevent Radiation-Induced SG Injury Amifostine (AMI) is the only FDA-approved radioprotective drug to prevent radiation-induced xerostomia. Although its protective mechanism remains unclear, theories suggest that AMI scavenges oxygen-derived free radicals. It is usually administered 30 min before IR IV at a dose of 200 mg/m2; however, it induces side effects like vomiting, nausea, and hypotension within 1 h of administration (King et al. 2020). Our laboratory is currently investigating whether the IG administration of AMI can provide comparable radioprotection to the SGs with reduced side effects. We plan to use the non-surgical transcutaneous IG injection technique to the submandibular SGs in mice to test AMI. The IG method of administering AMI will allow for more direct and less invasive access to the SGs, which could potentially limit side effects associated with the systemic administration. Also, AMI has shown to act synonymously with bone marrow stromal

A. A. Almansoori et al.

cells and suppress the effects of radiation, which could be explored in SGs as well using stem cell transplantation (Huang et al. 2019).

2.4

Safety and Efficacy of IG Injection

Initially, direct IG injection, using a surgical incision exposing the gland, was applied for various treatment modalities. The first study for BoNT injection into the parotids by Bushara et al. was performed using the surgical method but they noted that it could be associated with potential complications such as injury to the carotid artery or facial nerve branches (Bushara 1997). There was also the question of whether these injections could be well-tolerated in the pediatric population as well as the cost factor (Lakraj et al. 2013). Studies involving IG stem cell transplantations in mice also involved surgical incision into the mouse submandibular glands, and while they mention that it is relatively well-tolerated, they acknowledge that improved injection techniques may be useful (Lombaert et al. 2008). The introduction of ultrasound-guided IG injections reduced the invasiveness of the surgical technique. BoNT injections for the treatment of sialorrhea have been approved as a mainstay of treatment with ultrasound guidance, and studies have shown that it was well-tolerated in general (Teymoortash et al. 2007). Especially in the case of providing radioprotection, it was found that IG BoNT through ultrasound guidance did not interfere with the radio-chemotherapy treatment in patients (Teymoortash et al. 2007; Mueller et al. 2022). There were also minimal incidences of facial weakness or dysphagia, both of which could be seen with the surgical IG technique (Mueller et al. 2022). However, further larger studies assessing the efficacy of the ultrasound technique in gland preservation are required (Teymoortash et al. 2007). Ultrasound-guided IG injection to deliver stem cells is also gaining importance, and recent clinical trials are starting to show strong safety profiles, thus warranting larger studies

Drug Therapeutics Delivery to the Salivary Glands: Intraglandular and Intraductal Injections

to assess efficacy and feasibility (Lynggaard et al. 2022; Comella and Bell 2017; Grønhøj et al. 2017). It is still unclear as to whether the non-surgical IG technique is better performed with or without ultrasound guidance. So et al. demonstrated comparisons between the ultrasound- and nonultrasound-guided IG injections to determine which would be a safer and more accurate method (So et al. 2017). There was a significant difference in accuracy while injecting the parotid glands, with the ultrasound guidance being the more accurate method; however, there was no significant difference in accuracy while injecting the submandibular gland (So et al. 2017). Therefore, further studies confirming whether ultrasound guidance is required for IG injections are required, especially since ultrasound can be largely operator-dependent and expensive. Our laboratory will look to address this with the development of a novel non-surgical transcutaneous IG technique for the submandibular glands.

3

Intraductal (ID) Drug Delivery

Intraductal (ID) infusion of the salivary glands is regularly performed with sialography (Song and Lee 2014). During this imaging technique, contrast agents are taken up in the gland ducts and acini producing a parenchymal clouding (Schwalje and Hoffman 2019). A water-based contrast has been shown to reach the intercellular spaces and the basal surface of the acini cells and connective tissue (Qwarnström 1986). Su et al. evaluated the effective drug delivery route in the management of SG diseases using a porcine cadaver and methylene blue and found it was evenly distributed throughout the salivary gland (Su et al. 2017). The authors suggested that “intraductal injections might serve as a potential therapeutic procedure in the management of salivary gland disease.” (Schwalje and Hoffman 2019) Using this approach limits the use of systematic drugs with their inevitable side effect. It will also maximize the potential therapeutic effect by delivering a maximum dose to the gland only

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(Haroun and Brem 2000). The potential therapeutic application includes sialorrhea, sialadenitis, and Sjogren syndrome (Su et al. 2017).

3.1

Method

The ID infusion has been well performed for both parotid and submandibular salivary glands. Patients with active infection or inflammation are contraindicated from receiving this treatment. They are usually asked to attend well-hydrated and given a prophylactic antibiotic as indicated (Schwalje and Hoffman 2019). The patients are positioned semi-recumbent in preparation for the procedure (Schwalje and Hoffman 2019). For the parotid salivary gland, Stensen’s duct papillae are identified in humans opposite to the second upper molar using a salivary duct probe. For the submandibular gland, Wharton’s submandibular duct is identified in the floor of the mouth midway between the tongue ventral surface and lower incisors (Carlson 2000). Lidocaine-soaked gauze is applied to the duct papilla for topical anesthesia. The duct orifice is then dilated gradually with duct dilators (Su et al. 2017). A sialendoscopy might be used for proper viewing and drug infusion (Strychowsky et al. 2012). It is recommended to irrigate the gland with 1 ml of sterile saline or water before the drug infusion (Diggelmann and Hoffman 2015; Strychowsky et al. 2012). A 3 ml of the drug is then infused into the gland. A catheter might be introduced to keep the duct patent and left in place with a syringe attached for 2 min to prevent the immediate escape of the drug (Diggelmann and Hoffman 2015). Clinically, dilation of the duct orifice might be difficult in patients with chronic submandibular sialadenitis and chronic parotitis (Zenk et al. 2009). Additionally, repeated traumatic attempts of cannulization may raise the risk of orifice stricture (Antoniades et al. 2004). However, this difficulty might be solved with the use of sialoendoscopy. Initially, any mucus and stones should be removed along with dilation of the

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ductal stenosis. Secondly, the drug is then infused into the gland duct using the sialendoscope working channel. Lastly, a duct stent can be introduced and secured for weeks to enable repeated drug delivery and prevent re-stenosis (Su et al. 2016; Beilvert et al. 2014).

3.2

Type of Drugs and Therapeutic Applications

The first reported study was about the intraductal delivery of penicillin and saline for the treatment of chronic sialadenitis. Forty-four patients received penicillin and 11 patients were given saline. Among them, 22 patients were followed up and found to be symptom-free for an average of 11 years after the intraductal treatment (Su et al. 2017). Pilocarpine is one of the most used drugs for the treatment of xerostomia. It is given orally and might be associated with adverse side effects causing less patient compliance and tolerability to the drug (Wiseman and Faulds 1995; Berk 2008). The drug is also contraindicated in some medical conditions like chronic obstructive pulmonary disease and cardiovascular disease. The drug has a short duration of action (~4 h) and is required to be given two to three times daily. Therefore, locally administering pilocarpine into the gland with avoidance of systemic absorption will be much more beneficial. Muthumariappan et al. (2019) proposed a formulation consisting of pilocarpine-loaded poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) nanofiber mats. The outcomes of this in vitro study showed an initial release of pilocarpine at 26% (4.5 h), followed by a gradual increase (~46%) over 15 days. The pilocarpine-loaded nanofiber scaffold was compatible with the SG growth with normal cellular proliferation and homeostasis. Salivary secretion was significantly increased at 4.5 h after intradermal SG treatment with drugloaded nanofibers in vivo. After 24 h, no difference was noticed between these two treatment

formulations. Furthermore, the whole gland weight was comparable between the two formulations indicating no gross changes in SG composition and cellular content. Also, no histological differences were found between the treatment groups (Muthumariappan et al. 2019). IG injections of botulinum toxin (BTXA) are reliable for the treatment of sialorrhea (excessive saliva secretion) (Fuster Torres et al. 2007). On the other hand, a case report has shown the treatment of two patients with an ID infusion of BTXA (Schwalje and Hoffman 2019). Twentyfive units in 1 cc of reconstituted BTXA were infused into one of the major salivary ducts, followed by titration of up to 6 cc saline to the subject’s reported level of discomfort using a disposable pressure transducer (Schwalje and Hoffman 2019). Another recent study has also shown the use of intraductal corticosteroid infusion of triamcinolone for two patients with painful salivary swelling associated with Sjogren syndrome (Diggelmann and Hoffman 2015). In one patient, she was symptom-free for 2 months at the time of follow-up, and for the other patient, symptoms recurred 3 months later. The treatment was repeated three times and the patient remained symptom-free for 8 months since the last injection (Diggelmann and Hoffman 2015). ID infusion of the salivary gland might not only be limited to medication. Cell-based therapies have been tried for the treatment of atrophic salivary glands (Almansoori et al. 2019). Recently, Kasamatsu et al. have evaluated the intraductal delivery of salivary cells for irradiated glands. They cultured rat salivary gland cells in a medium with Rho kinase inhibitor (RI) and then transplanted them via the duct into the submandibular glands of irradiated atrophic SG rats. Twelve weeks after the transplantation, immunohistochemical analysis was performed, and salivary flow rates were measured (Kasamatsu et al. 2022). The cells were found to be located in the ductal region following transplantation and alpha-amylase (AMY) expression,

Drug Therapeutics Delivery to the Salivary Glands: Intraglandular and Intraductal Injections

and the salivary flow was higher in the salivary cell transplantation group. The results showed that intraductal cell-based therapy using RI-treated SG cells was able to restore salivary secretion of the irradiated salivary gland. They suggested this therapy can be applied clinically by culturing the labial minor salivary gland cells of patients before the radiotherapy and transplanting them through the salivary gland duct later. The same therapy can be used for hyposalivation associated with aging and Sjogren syndrome (Kasamatsu et al. 2022). In our lab, we have been using the intraductal route for decellularization and recellularization of the rat submandibular salivary gland. The purpose of this approach is to produce a bioengineered salivary gland to be used for transplantation purposes in absence of immune rejection. For decellularization, the submandibular salivary gland was harvested along with its vascular pedicles (linguofacial vein and carotid artery) and duct. The gland was flushed for 15 min using saline that contains 50 U/mL heparin. Two 26-gauge catheters were inserted into the gland artery, and duct, and fixed with 8/0 silk sutures. The gland was decellularized by infusion with 10% sodium dodecyl sulfate (SDS) for 2 h at room temperature. The gland was then infused through the duct with 1%Triton-X100 for 1 h at room temperature. The gland was then treated with benzonase (90 U/mL) for 60 min to remove remnant nuclei. Subsequently, the gland was rinsed with phosphatebuffered saline containing penicillin (100 U/mL), streptomycin (100 U/mL), and amphotericin B (2.5 mg/L) for 30 min and stored at 4 °C. Recellularization was performed as mentioned in previous studies (Doi et al. 2017; Uygun et al. 2010; Gao et al. 2014). Briefly, the decellularized gland was soaked in phosphate-buffered saline (PBS) containing 1% penicillin-streptomycinamphotericin at 4 °C for 1 h. The gland was then equilibrated in the cell culture medium for 3 h. A density of 13 × 106 immortalized human salivary gland acinar cells (NSSVAC) were

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seeded via the salivary gland duct. The cells were injected at a rate of 1 ml/min at four steps, 2 ml/step, with 10 min intervals between each step. The overnight static culture was conducted to allow the cell’s attachment. The next day, the medium was perfused through the gland artery at a 0.5 ml/min rate while the gland is soaked within a culture medium in the glass jar. The whole system was placed in the incubator at 37 °C with 5% CO2 and 95% O2. The perfusion rate was changed to 4 ml/min from the second day for 14 days culture period. The medium was changed every 48 h. Our primary results showed complete decellularization using the intraductal route shown in the hematoxylin and eosin (H&E) staining and DNA quantification assessment with preservation of the acinar and duct structures and removal of the cellular components. The decellularized salivary gland also preserved the collagen fibers well as shown by the trichrome stain and total collagen assay. The recellularized gland showed a grossly homogenous distribution of the seeded cells, and the H&E staining showed the cells well-attached and grown inside the gland (Fig. 2).

3.3

Efficiency and Limitations

The abovementioned studies have supported the use of intraductal injections as potential therapeutic approaches for the treatment of salivary gland diseases. They demonstrated the utility of intraductal infusion of penicillin, botulinum toxin, corticosteroid, and salivary cells for the treatment of sialadenitis, hypersalivation, Sjogren syndrome, and irradiated salivary glands. The intraductal approach is free of systemic side effects and ensures a maximum dose delivery to the salivary gland. It also decreases the risks of percutaneous needle injection. These studies have also provided evidence for the safety of intraductal salivary gland infusion.

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Fig. 2 Stages of decellularization and recellularization of rat SMG through intraductal route. (a) Two 26-gauge catheters were placed into the gland duct and artery. (b) White translucent SMG scaffold after completing the decellularization. (c) SMG scaffold during recellularization.

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(d) Brown-look SMG after recellularization. (e) Trichrome staining showing the preservation of collagen fibers and the absence of cellular content. (f) H&E staining showing the attachment of seeded salivary gland acinar cells

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Drug Therapeutics Delivery to the Salivary Glands: Intraglandular and Intraductal Injections organ decellularized bioscaffold. Cells Tissues Organs 200(3–4):171–180 Grønhøj C, Jensen DH, Glovinski PV, Jensen SB, Bardow A, Oliveri RS et al (2017) First-in-man mesenchymal stem cells for radiation-induced xerostomia (MESRIX): study protocol for a randomized controlled trial. Trials 18(1):e108 Haroun RI, Brem H (2000) Local drug delivery. Curr Opin Oncol 12(3):187–193 Huang B, He T, Yao Q, Zhang L, Yao Y, Tang H et al (2019) Amifostine suppresses the side effects of radiation on BMSCs by promoting cell proliferation and reducing ROS production. Stem Cells Int 2019: 8749090 Isabelle S, Li L, Veraragavan A, Robert et al (2013) Combined KIT and FGFR2b signaling regulates epithelial progenitor expansion during organogenesis. Stem Cell Rep 1(6):604–619 Jost WH, Friedman A, Michel O, Oehlwein C, Slawek J, Bogucki A et al (2019) SIAXI. Neurology 92(17): e1982–e1991 Kasamatsu A, Fukushima R, Nakamura K, Kawasaki K, Yoshimura S, Koyama T et al (2022) A novel cellbased transplantation method using a Rho kinase inhibitor and a specific catheter device for the treatment of salivary gland damage after head and neck radiotherapy. Biochem Biophys Rep 32:101385 Kim JW, Kim JM, Choi ME, Kim S-K, Kim Y-M, Choi J-S (2019) Adipose-derived mesenchymal stem cells regenerate radioiodine-induced salivary gland damage in a murine model. Sci Rep 9(1):15752 King M, Joseph S, Albert A, Thomas TV, Nittala MR, Woods WC et al (2020) Use of amifostine for cytoprotection during radiation therapy: a review. Oncology 98(2):61–80 Lakraj A, Moghimi N, Jabbari B (2013) Sialorrhea: anatomy, pathophysiology and treatment with emphasis on the role of botulinum toxins. Toxins 5(5):1010–1031 Lim JY, Yi T, Choi JS, Jang YH, Lee S, Kim HJ et al (2013) Intraglandular transplantation of bone marrowderived clonal mesenchymal stem cells for amelioration of post-irradiation salivary gland damage. Oral Oncol 49(2):136–143 Lombaert IMA, Brunsting JF, Wierenga PK, Faber H, Stokman MA, Kok T et al (2008) Rescue of salivary gland function after stem cell transplantation in irradiated glands. PLoS One 3(4):e2063 Lynggaard CD, Grønhøj C, Christensen R, FischerNielsen A, Melchiors J, Specht L et al (2022) Intraglandular off-the-shelf allogeneic mesenchymal stem cell treatment in patients with radiation-induced xerostomia: a safety study (MESRIX-II). Stem Cells Transl Med 11(5):478–489 Mohamed NH, Shawkat S, Moussa MS, Ahmed N (2022) Regeneration potential of bone marrow derived mesenchymal stem cells and platelet rich plasma (PRP) on irradiation-induced damage of submandibular salivary gland in albino rats. Tissue Cell 76:101780

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Mueller J, Langbein T, Mishra A, Baum RP (2022) Safety of high-dose botulinum toxin injections for parotid and submandibular gland radioprotection. Toxins 14(1):64 Muthumariappan S, Ng WC, Adine C, Ng KK, Davoodi P, Wang C-H et al (2019) Localized delivery of pilocarpine to hypofunctional salivary glands through electrospun nanofiber mats: an ex vivo and in vivo study. Int J Mol Sci 20(3):541 Nanduri LSY, Lombaert IMA, Van Der Zwaag M, Faber H, Brunsting JF, Van Os RP et al (2013) Salisphere derived c-Kit+ cell transplantation restores tissue homeostasis in irradiated salivary gland. Radiother Oncol 108(3):458–463 Narmada IB, Laksono V, Nugraha AP, Ernawati DS, Winias S, Prahasanti C et al (2019) Regeneration of salivary gland defects of diabetic wistar rats post human dental pulp stem cells intraglandular transplantation on acinar cell vacuolization and interleukin-10 serum level. Pesquisa Brasileira em Odontopediatria e Clínica Integrada 19(1):1–10 Nigam PK, Nigam A (2021) Botulinum toxin. Indian J Dermatol 55:8–14 Ogle OE (2020) Salivary gland diseases. Dental Clin 64(1):87–104 Pal PK, Calne DB, Calne S, Tsui JKC (2000) Botulinum toxin A as treatment for drooling saliva in PD. Neurology 54(1):244–244 Qwarnström E (1986) Experimental sialography: the effects of retrograde infusion of radiographic contrast media on salivary gland morphology and function: a review article. Oral Surg Oral Med Oral Pathol 62(6): 668–682 Rabea AA, Rashed L, Hassan R (2022) Regenerative capacity of bone marrow stem cells on aged albino rat’s parotid excretory duct. Arch Oral Biol 141: 105470 Schwalje AT, Hoffman HT (2019) Intraductal salivary gland infusion with botulinum toxin. Laryngoscope Investig Otolaryngol 4(5):520–525 Schwarz S, Huss R, Schulz-Siegmund M, Vogel B, Brandau S, Lang S et al (2014) Bone marrow-derived mesenchymal stem cells migrate to healthy and damaged salivary glands following stem cell infusion. Int J Oral Sci 6(3):154–161 Scott AB (1981) Botulinum toxin injection of eye muscles to correct strabismus. Trans Am Ophthalmol Soc 79: 734–770 Sherif H, Esmail A, Abdelfattah M, Liu Z-J, Abouzid W (2018) Evaluation of the effects of Botulinum Neuro Toxin A (BoNTA) on submandibular salivary gland in rats by histological examination and semi-quantitative scoring method. Al-Azhar J Dental Sci 21(5):511–518 So JI, Song DH, Park JH, Choi E, Yoon JY, Yoo Y et al (2017) Accuracy of ultrasound-guided and nonultrasound-guided botulinum toxin injection into cadaver salivary glands. Ann Rehabil Med 41(1):51 Song GG, Lee YH (2014) Diagnostic accuracies of sialography and salivary ultrasonography in Sjögren’s

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Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 131–152 https://doi.org/10.1007/5584_2023_773 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 Published online: 16 March 2023

6

Negative-Pressure Wound Therapy: What We Know and What We Need to Know

7 8 9

Toshifumi Yamashiro , Toshihiro Kushibiki , Yoshine Mayumi , Masato Tsuchiya , Miya Ishihara and Ryuichi Azuma

10 11 12

,

13 14

Abstract

15

Negative-pressure wound therapy (NPWT) promotes wound healing by applying negative pressure to the wound surface. A quarter of a century after its introduction, NPWT has been used in various clinical conditions, although molecular biological evidence is insufficient due to delay in basic research. Here, we have summarized the history of NPWT, its mechanism of action, what is currently known about it, and what is expected to be known in the future. Particularly, attention has shifted from the four main mechanisms of NPWT to the accompanying secondary effects, such as effects on various cells, bacteria, and surgical wounds. This chapter will help the reader to understand the current status and shortcomings of NPWT-related research, which could aid in the development of basic research and,

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

T. Yamashiro, M. Tsuchiya, and R. Azuma Department of Plastic and Reconstructive Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan e-mail: [email protected]; [email protected]; [email protected] T. Kushibiki (✉), Y. Mayumi, and M. Ishihara Department of Medical Engineering, National Defense Medical College, Tokorozawa, Saitama, Japan e-mail: [email protected]; [email protected]; [email protected]

eventually, clinical use with stronger scientific evidence.

33

Keywords

35

Angiogenesis · Biofilm · Epithelialmesenchymal transition · Mechanotransduction · Microdeformational wound therapy · Surgical site infection · Topical negative pressure · Vacuum-assisted closure · Vacuum sealing drainage · Wound healing

36

34

37 38 39 40 41 42

Abbreviations

43

ALP Ang-1 Ang-2 BDNF bFGF EGF IL iNPWT

44

MAPK MDSC MMP NPWT

alkaline phosphatase angiogenin-1 angiogenin-2 brain-derived neurotrophic factor basic fibroblast growth factor epidermal growth factor interleukin incisional negative-pressure wound therapy mitogen-activated protein kinase muscle-derived stem cell matrix metalloproteinase negative-pressure wound therapy 131

45 46 47 48 49 50 51 52 53 54 55 56 57

132 58

NPWTi-d

59 60

P-MSC

61 62

sNPWT

63 64 65 66 67 68

69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102

T. Yamashiro et al.

SSI TGF-β TNF-α VEGF α-SMA

1

negative-pressure wound therapy with instillation and dwell time periosteum-derived mesenchymal stem cell single-use negative-pressure wound therapy surgical site infection transforming growth factor-beta tumor necrosis factor-alpha vascular endothelial growth factor smooth muscle actin α

Introduction

Since the initial introduction of negative-pressure wound therapy (NPWT) in 1997 (Morykwas et al. 1997), this innovative treatment has been widely adopted in difficult-to-treat wounds, primarily those of the skin and soft tissue (Poteet et al. 2021). This approach, in which the wound surface is sealed with gauze, polyurethane foam, or other materials and a film dressing, followed by internal suction, is also called topical negative pressure (TNP), vacuum sealing drainage (VSD), or microdeformational wound therapy (MDWT) and is known to promote wound healing through various mechanisms. It is recognized as a clinically useful treatment in many areas of practice, including emergency, abdominal and thoracic surgery, orthopedic surgery, and plastic surgery. Specifically, clinical studies have shown its effectiveness in treating difficult-to-treat wounds, including diabetic foot lesions (Meloni et al. 2015; Chen et al. 2021); postoperative wound dehiscence (Seidel et al. 2020) and sternal osteomyelitis (Steingrimsson et al. 2012); trauma wounds, including burns (Kantak et al. 2016); skin and soft tissue defects resulting from resection of benign and malignant tumors (Fourman et al. 2022); and infected wounds (Faust et al. 2021) as well as preventing complications following primary closure surgery (Norman et al. 2022). Although a quarter of a century has passed since its introduction, the molecular biological mechanism involved in NPWT remains unclear, and the level of evidence for its effectiveness remains

insufficient, despite clinicians’ reassurances of its effectiveness. This is partly due to the lack of basic research, including in vitro and in vivo studies, compared to the many clinical reports. Additionally, the complexity of the healing mechanism of NPWT and the difficulty of reproducing it in basic research may be one of the reasons for the small number of studies and low level of evidence. An increase in the level of evidence would greatly contribute to expanding the range of indications for NPWT by providing new therapeutic targets and reducing complications. In this chapter, we first describe the history of NPWT and its status in clinical practice, followed by the molecular biological mechanisms currently known and areas expected to be elucidated in the future.

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History and Variations of NPWT

Commercial negative-pressure therapy devices were available in 1995, and in 1997. Morykwas et al. (1997) first reported a novel treatment, later called NPWT. They reported that -125-mmHg negative pressure caused increased wound granulation and periwound blood flow in a porcine model. Argenta and Morykwas (1997) reported its clinical usefulness in a case study of 300 patients. Their treatment involved applying a porous sponge to the wound surface, sealing it with a film dressing, and applying constant negative pressure with a specialized treatment device. This new treatment method was rapidly accepted throughout the 2000s, and in the 2010s, various treatment variations appeared with equipment development. Unlike conventional NPWT, which involves a foam and film contact surface, single-use NPWT (sNPWT) (Fong and Marston 2012; Sharp 2013; van den Bulck et al. 2013) comprises a special dressing that integrates a nonadherent contact surface and a sponge portion that absorbs the exudate. Many of these devices are battery-operated and disposable; thus, they are portable and can be used in outpatients. Furthermore, sNPWT was found to be more effective than regular NPWT in patients with

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diabetic foot lesions (Kirsner et al. 2021). Recent reports indicate that sNPWT effectively prevents wound infection in primary closed wounds after abdominal surgery (Norman et al. 2022). Intermittent NPWT or variable NPWT (Borgquist et al. 2010a; Lee et al. 2015), in which the intensity of negative pressure is varied periodically, is said to be superior to continuous negative pressure in increasing periwound blood flow (Borgquist et al. 2010a; Sogorski et al. 2018). This was already pointed out by Morykwas et al. (1997) in their first report; however, only continuous negative pressure was widely known owing to the capability of the treatment device, and intermittent NPWT was brought back into focus only around 2010 (Ahearn 2009). Despite its superior therapeutic effect, it has been noted that patient discomfort is greater with intermittent NPWT than with continuous negative pressure due to repetitive pain at the start of each cycle (Borgquist et al. 2010a; Malmsjo et al. 2012). A treatment combining NPWT with wound irrigation has been considered since the early 2000s (Wolvos 2004). A typical example is NPWT with instillation and dwell time (NPWTid), which has become easier to use since 2011, when a device with an improved irrigation system (from gravity to pump) was launched into the market. This treatment is based on the idea that the wound can be cleaned by repeating the cycle of injecting the cleaning solution into the wound, immersing it for a certain period, and then suctioning it under negative pressure (Wolvos 2013), making it possible to apply NPWT to wounds with infection (Kanapathy et al. 2020). NPWT is also used for dressing open abdominal wounds that cannot be closed primarily due to intestinal edema or other conditions that increase the risk of abdominal compartment syndrome (Bjarnason et al. 2011; Bertelsen et al. 2014). However, its indication, safety, and comparison to other treatments are still under debate (Cheng et al. 2022). All these treatment variations have gained wide acceptance in clinical practice. Thus, NPWT has evolved along with the development of commercial negative-pressure treatment devices. Although its usefulness is widely

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recognized in many clinical studies, only few basic research reports have examined its treatment mechanism in detail.

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Principal Mechanisms of NPWT

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The effects of NPWT in promoting wound healing are thought to involve four primary mechanisms and their accompanying secondary effects (Orgill et al. 2009; Huang et al. 2014; Normandin et al. 2021) (Fig. 1). The primary mechanisms are (1) coarse wound contraction (macrodeformation), (2) microscopic deformation of the wound surface (microdeformation), (3) fluid removal, and (4) stabilization of the wound environment (Fig. 2). The interaction of these mechanisms results in various secondary effects.

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When a wound is filled with foam, such as gauze or porous sponge, covered with a film dressing, and negative pressure is applied, the wound shrinks as the foam shrinks. In experiments with polyurethane foam by Scherer et al. (2008), the negative pressure of -125 mmHg reduced the volume of the foam by approximately 80%. This gross contraction effect depends on the nature and mobility of the tissue surrounding the treatment site, suction pressure, and material and volume of the foam (Orgill et al. 2009; Borgquist et al. 2011; Anesater et al. 2011). The contraction effect is stronger in areas with thick subcutaneous fat, loose skin, and high flexibility, such as the abdominal wall, and less effective in areas with low flexibility, such as the scalp and scar-covered wounds (Orgill et al. 2009). This is supported by reports that subcutaneous fat exhibits a stronger contractile response than skin at the same site (Torbrand et al. 2010; Katzengold et al. 2018). With the wound shrinkage, the wound area is physically reduced due to edema reduction, granulation, and scarring (Torbrand et al. 2010; Katzengold et al. 2018; Borgquist et al. 2010b).

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Fig. 1 Mechanisms of action of NPWT. Negative-pressure wound therapy (NPWT) promotes wound healing through four primary mechanisms and associated secondary effects

Fig. 2 Schematic illustration of NPWT. Negativepressure wound therapy (NPWT) is associated with four primary mechanisms of action: (1) macrodeformation,

(2) microdeformation, (3) fluid removal, and (4) stabilization of the wound environment

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Mechanical loading of the tissue causes deformation (McLeod et al. 1987). In NPWT, the pressure on the wound surface increases in contrast to the low-pressure environment inside the dressing (Kairinos et al. 2009a, b; Biermann et al. 2020;

Sogorski et al. 2022). The minute tissue deformation at the foam-wound boundary resulting from this is called microdeformation (Borgquist et al. 2010b; Saxena et al. 2004). This mechanical force propagates into the tissue through the extracellular fluid and produces shear and deformation forces on individual cells (Orgill et al. 2009;

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Wilkes et al. 2009a; Lu et al. 2011). Finite element studies have shown that under typical treatment conditions using porous sponges, approximately 20% strain is exerted on the wound surface (Saxena et al. 2004; Wilkes et al. 2009a, b). In response to this mechanical stimulation, effects, such as increased cell proliferation and migration, angiogenesis, and granulation, are observed (Saxena et al. 2004; Hsu et al. 2010; Toume et al. 2017). The process by which cells sense mechanical stimuli and convert them into physiological responses and gene expression is called mechanotransduction. It is believed to occur primarily in the cytoskeleton via mechanoreceptors, such as integrin β1 (Wang et al. 1993; Huang et al. 1998). Mechanotransduction is often mentioned in studies of fibroproliferative diseases characterized by excessive connective tissue accumulation and persistent tissue contraction, and signaling pathways, such as transforming growth factor-β (TGF-β)/Smad, mitogen-activated protein kinase (MAPK), Rho/ROCK, Wnt/β-catenin, and tumor necrosis factor-α (TNF-α)/nuclear factorkappa B (NF-κB), are known to be involved (Huang and Ogawa 2012). For instance, in skin fibroblasts, integrins act as mechanical sensors for bidirectional signaling between cells and the extracellular matrix (ECM) (Katsumi et al. 2004). The ECM transduces mechanical stimuli to the cytoskeleton via the integrin-focal adhesion kinase (FAK) pathway (Santos and Lagares 2018), and ECM stiffness affects mechanical signaling through the TGF-β1 pathway. Integrins activated by the stiff ECM stimulate TGF-β1 release and bind to the TGF-β1 receptor on myofibroblasts. This induces a positive feedback that increases TGF-β1 and smooth muscle actin α (α-SMA) levels and stiffens the ECM. Conversely, a soft ECM suppresses TGF-β1 and α-SMA expression and further softens the ECM (Santos and Lagares 2018; Fu et al. 2021). In keratinocytes, mechanical stress-induced proliferative changes occur via mechanisms such as the ECM-integrin pathway, MAPK pathway, and epithelial-mesenchymal interactions (Reichelt 2007), while in endothelial cells, mechanical stimulation is known to cause angiogenesis and vascular remodeling (Fu et al.

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2021). NPWT-induced cellular responses, such as increased cell proliferation and migration as well as changes in transcription factors associated with wound healing, are thought to be mainly the result of microdeformation and mechanotransduction induced by mechanical stimuli. For this reason, microdeformation has been considered the most important of the four main mechanisms (Orgill et al. 2009; Huang et al. 2014; Normandin et al. 2021; Wiegand and White 2013).

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Fluid Removal

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NPWT drains the exudate from the wound surface, and the pressure gradient allows the extracellular fluid in the tissue to discharge. This reduces tissue edema (Orgill et al. 2009; Labanaris et al. 2009), relieves microvascular compression caused by excess interstitial fluid, and may contribute to increased blood flow to the wound (Huang et al. 2014). The amount of exudate depends on the condition of the wound; thus, appropriate pressure settings are necessary (Borgquist et al. 2011). Wound exudate contains inflammatory cytokines and proteolytic enzymes that inhibit wound healing, such as TNF-α and matrix metalloproteinase (MMP). It is thought that NPWT promotes wound healing not only by promoting cell proliferation but also by eliminating and controlling these factors along with the exudate (Stechmiller et al. 2006; Moues et al. 2008; Glass et al. 2014).

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Stabilization of the Wound Environment

In NPWT, dressings are made with a semipermeable polyurethane film to ensure airtightness in addition to the foam material. This partially restricts gas exchange and water vapor permeation, which provides heat and moisture retention and is beneficial for wound healing (Orgill et al. 2009; Kloth et al. 2002; Winter and Scales 1963; Hinman and Maibach 1963). The dressing is also impermeable to proteins and bacteria and acts as a

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physical barrier to the outside world (Orgill et al. 2009; Huang et al. 2014). Materials used for the foam in direct contact with the wound include polyurethane and polyvinyl alcohol. However, noncommercial alternatives, such as those using gauze and wall suction, are also used owing to cost concerns (Bui et al. 2006; Gibson 2022). Veerasubramanian et al. (2021) investigated the function of the foam material itself on the inflammatory response of macrophages in vitro and reported that it had an inhibitory effect on the inflammatory molecules TNF-α and interleukin1β (IL-1β). It has also been noted that a decrease in partial pressure of oxygen in the dressing during negative-pressure treatment may affect bacterial activity (Biermann et al. 2019), suggesting that the therapeutic effect of NPWT is not solely due to mechanical stimulation.

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The four primary mechanisms described above interact to produce cellular responses and secondary effects that lead to wound healing (Fig. 1). In this section, we discuss the cellular response, immune responses, angiogenesis, and granulation, which have received particular attention, as well as the effects on epithelialization, osteogenesis, tumors, and bioburden and the latest findings on the effects on surgical procedures, such as primary closed wounds, skin grafts, and flap operation.

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Secondary Effects of NPWT

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As mentioned previously, mechanical stimulation with NPWT alters cell function through microdeformation and mechanotransduction. Cytoskeletal changes affect cell function and, consequently, tissue function (Folkman and Moscona 1978; Alford et al. 2011); more highly distorted cells are more sensitive to soluble mitogens and promote cell proliferation (Chen et al. 1997; Ingber 2005). Saxena et al. (2004) reported, via finite element method simulations, that the 5–20% strain produced by NPWT was equivalent to the strain level that promotes cell proliferation

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Cellular Responses

in vitro. Toume et al. (2017) and Katzengold et al. (2021) confirmed that wound closure was accelerated in experiments where elongation forces equivalent to those during NPWT treatment were applied directly to cultured fibroblast wound models. Their studies mainly focused on strain, among the various complex therapeutic mechanisms of NPWT. Multiple experiments in mice showing that NPWT increased the expression of Ki-67, a marker of cell proliferative potential (Scherer et al. 2009; Dastouri et al. 2011; Shao et al. 2021), corroborate the biological effects. Growth factors, binding to ECM proteins, and isometric tension are required for cells to proliferate and differentiate effectively (Ingber 2005; Huang and Ingber 1999). In chronic wounds, the cell scaffold is unstable, resulting in inadequate isometric tension among these three factors, which prevents cell proliferation and differentiation. Mechanical stimulation with NPWT may compensate for this lack of isometric tension and further promote wound healing by affecting the mechanical environment within the tissue (Huang et al. 2014).

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Immune Responses

As mentioned in Sect. 3.3, NPWT contributes to the regulation of wound inflammation by removing exudate containing inflammatory substances. In patients with traumatic soft tissue injuries treated with NPWT or a bilayer wound dressing made of Teflon and polyurethane, the amount of IL-8 in the exudate was found to be significantly higher in the NPWT group (Labler et al. 2009). In addition, biopsy experiments examining gene expression in human split-thickness skin wounds showed increased expression of leukocyte chemoattractants, such as IL-8 and CXCL5 (Nuutila et al. 2013). IL-8, a potent chemokine and angiogenesis-promoting factor, plays an important role in regulating the migration of neutrophils and macrophages (Koch et al. 1992; Mukaida et al. 1998). These findings indicate that NPWT may affect the inflammatory phase of wound healing by removing soluble chemokines and infiltrating leukocytes along with exudate (Glass et al. 2014).

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4.3

Angiogenesis and Tissue Blood Flow

NPWT creates a hypoxic environment outside the wound (Biermann et al. 2019), and microdeformation also causes temporary localized hypoperfusion and hypoxia at the wound margins (Erba et al. 2011). Hypoxia increases vascular endothelial growth factor (VEGF) expression via the upregulation of hypoxia-inducible factor-1α (HIF-1α). This hypoxia and VEGF expression contribute to directional, more near-physiologic angiogenesis because it exhibits the strongest concentration gradient at the wound edge (Erba et al. 2011) (Fig. 3). Thus, NPWT increases microvascular density at the treatment site and increases wound blood flow (Greene et al. 2006;

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Malsiner et al. 2015). Ma et al. (2016) focused on changes in growth factors over time in an in vivo study involving patients. They reported that NPWT increased the expression of angiogenin-2 (Ang-2) and decreased the expression of angiogenin-1 (Ang-1) and the Ang-1/ Ang-2 ratio in the early phase of treatment; meanwhile, it increased the expression of Ang-1, Ang-1/Ang-2 ratio, and phosphorylation level of the tyrosine kinase receptor Tie-2 in the late phase. This implies that NPWT increases angiogenesis by stimulating microvascular destabilization and regression in the early stages of treatment and promotes microvascular maturation by prioritizing microvascular stabilization in the later stages (Ma et al. 2016). They also demonstrated that pericytes and collagen IV are involved

Compression force Relative low pressure inside dressing

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Fig. 3 Angiogenesis induced by NPWT. The hypoxic environment inside and outside the wound caused by negative-pressure wound therapy (NPWT) activates the

HIF-1α-VEGF pathway, leading to angiogenesis. HIF-1α hypoxia-inducible factor-1α, VEGF vascular endothelial growth factor

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in the maturation of microvessels in the late stages of NPWT using a rat diabetic wound model (Ma et al. 2017). The effect of NPWT on tissue blood flow has long been a subject of debate. Morykwas et al. (1997) first reported that NPWT increases wound edge blood flow via evaluation with laser Doppler. Sogorski et al. performed experiments on healthy volunteers. They showed that the blood flow of their normal thigh skin under NPWT increased by up to +151% and tissue oxygen saturation increased by up to +28.2%, noting that this effect may be greater with variable negative pressure than with continuous negative pressure (Sogorski et al. 2018, 2022). Conversely, Wackenfors et al. (2004) reported that changes in blood flow were affected by the distance from the wound margin, with blood flow reduced at positions very close to the wound. Their results in porcine models are supported by in vitro simulation experiments (Biermann et al. 2020) and experiments that evaluated radioisotope perfusion and transcutaneous oxygen saturation in healthy volunteers (Kairinos et al. 2009b). Therefore, it is believed that, in NPWT, attention should be paid to the possibility of adverse effects on tissue blood flow when applied to ischemic lesions or when used circumferentially on the limb (Kairinos et al. 2009b; Livingstone et al. 2021; Singh et al. 2021). When considering these contradictory results regarding the effect of NPWT on tissue blood flow, it is necessary to focus on the timing of the blood flow measurements. When discussing NPWT today, it is generally considered that NPWT promotes angiogenesis and increases blood flow to the wound in the long term (Panayi et al. 2017).

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Granulation tissue in wounds is specialized for wound healing and is associated with cellular findings, such as the proliferation of vascular endothelial cells, pericytes, fibroblasts, and myofibroblasts, and mobilization of macrophages (Diaz-Flores Jr. et al. 2009). Morykwas and Argenta first reported on the promotive effect

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Granulation Tissue Formation

of NPWT on granulation growth (Morykwas et al. 1997; Argenta and Morykwas 1997). This promotive effect on granulation growth has been investigated mainly through cell experiments using bioreactors that reproduce the NPWT environment in vitro. Wilkes et al. (2007) successfully reproduced a treatment environment similar to that of commercial devices by applying a negative-pressure load on 3D-cultured fibroblasts via foam dressing using 6-well plates and cell inserts. They observed that a 48-h treatment caused fibroblasts to thicken and become elongated and bipolar. Similarly, experiments with 3D-cultured fibroblasts have confirmed that NPWT improves the energetic state of fibroblasts by increasing adenosine triphosphate (ATP) and cytochrome c oxidase levels, significantly increasing TGF-β and platelet-derived growth factor (PDGF) α,β levels (McNulty et al. 2007; McNulty et al. 2009), and upregulating the expression of the messenger RNA of type 1 collagen α1 (COL1A1), basic fibroblast growth factor (bFGF), TGFβ1, and α-SMA (Lu et al. 2011). All these findings indicate the upregulation of collagen production, which is the basis for promoting granulation growth. Furthermore, experiments using mast cell-deficient mice have shown that NPWT induces wound tissue granulation, cell proliferation, and angiogenesis during the proliferative phase of wound healing and that mouse mast cell proteases 4, 5, and 6 (mMCP 4, 5, 6) in the secretory granules of mast cells may play an important role in this process (Younan et al. 2011; Succar et al. 2014).

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Reepithelialization and Epithelial-Mesenchymal Transition

Transcriptome analysis in patients has shown that NPWT may promote reepithelialization during the wound inflammation phase by increasing epithelial cell migration and proliferation but may impair epidermal maturation in the long term by reducing differentiation (Nuutila et al. 2013). Gene expression profiling of 7-day

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NPWT-adapted split-thickness skin graft donor site wounds showed that the most induced genes encoded ILs (IL8, IL24), prostaglandins (COX2), chemokines (CXCL5), osteostatins (parathyroid hormone-like hormone (PTHLH)), and MMPs (MMP1, MMP3, and MMP10), which revealed that factors associated with cell proliferation and inflammation were induced (Nuutila et al. 2013). Furthermore, genes particularly suppressed were those related to the formation of the cornified envelope (CE), which consists of stratified squamous cells and acts as a protective barrier to the epidermis (Sun and Green 1976) (LOR, FLG, annexin A9 (ANX9), and LCE) and cytokeratins (KRT1, KRT2, and KRT10), suggesting that the development and maturation of the neoepidermis may be disturbed (Nuutila et al. 2013). Hsu et al. reported the results of a series of notable in vitro studies in which the behavior of keratinocytes in monolayer culture was analyzed using electrical cell-substrate impedance sensing (ECIS) methods and morphological observations (Hsu et al. 2010, 2013; Chow et al. 2016; Huang et al. 2016). A negative-pressure environment of -125 mmHg stimulated the appearance of cell division control protein 42 (Cdc42) at the leading edge of epithelial cells, which may facilitate epithelial cell migration by promoting actin polymerization and the formation of lamellipodia and filopodia (Hsu et al. 2010, 2013). They also found that negative-pressure treatment decreased the expression level of β-catenin in human keratinocytes at the plasma membrane, while it increased in the nucleus with elevated activity. They reported that Src-dependent phosphorylation of p120-catenin (p120ctn) at the plasma membrane caused a downregulation of the adhesion molecule E-cadherin followed by degradation of the adherens junction (Chow et al. 2016; Huang et al. 2016). Liu et al. (2022) also reported that negative pressure in vitro downregulates miR-203 expression and promotes cell proliferation and migration of human keratinocytes via upregulation of the p63 protein. We monitored human keratinocytes during negative-pressure treatment in real time and found that migration was enhanced under continuous and intermittent negative pressure (Yamashiro et al. 2022). In

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summary, these reports suggest that NPWT contributes to wound healing by weakening the intercellular junctions of epithelial cells and promoting cell movement. This biomarker change in keratinocytes by NPWT recalls an association with epithelialmesenchymal transition (EMT) (Zeisberg and Neilson 2009) (Fig. 4). EMT is a phenomenon in which epithelial cells acquire mesenchymal cell-like characteristics and is thought to be involved in embryonic development, fibrosis, wound healing, and cancer progression (Nieto et al. 2016; Dongre and Weinberg 2019). As a result of EMT, epithelial cells lose their cell polarity and cell adhesive function with surrounding cells and gain migration and invasive capacity, transforming into mesenchymal-like cells (Nieto et al. 2016; Dongre and Weinberg 2019) (Fig. 4). EMT-promoting factors include TGF-β, bFGF, epidermal growth factor (EGF), and hepatocyte growth factor (HGF) (Sistigu et al. 2017; Koike et al. 2020). EMT in wound healing may play an important role in reepithelialization from the wound margin (Haensel and Dai 2018). Despite these clues, the association between NPWT and EMT has not been examined. An in vitro study showed that EMT was promoted by intermittent negative pressure on triple-negative breast cancer cells (Liu et al. 2018a). EMT is also induced in human keratinocytes cultured in a microgravity environment through a decrease in levels of the epithelial marker E-cadherin; increase in the levels of mesenchymal markers, such as vimentin and α-SMA, as well as typical EMT transcription factors, such as Snail1, Snail2, and ZEB2; and increase in the levels of MMPs (Ranieri et al. 2017). These findings may lead to a discussion of the potential of EMT as a mechanism of action for NPWT.

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Ilizarov reported that osteogenesis can occur following the application of a sustained elongation force to the bone (Ilizarov 1989a, b); bone lengthening is one of the most common examples of mechanotransduction being incorporated into

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EMT Epithelial

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Fig. 4 Possible association of NPWT with EMT. Epithelial-mesenchymal transition (EMT) refers to the process by which epithelial cells acquire mesenchymal cell-like properties. EMT is a dynamic and transitional state between epithelial and mesenchymal phenotypes, and the opposing process is referred to as mesenchymalepithelial transition (MET). The epithelial cell changes

induced by negative-pressure wound therapy (NPWT) are similar to those observed in the process of EMT, and the same may apply to its effects on tumor cells. EMT epithelial-mesenchymal transition, MET mesenchymalepithelial transition, NPWT negative-pressure wound therapy, α-SMA smooth muscle actin α, MMP matrix metalloproteinase

clinical practice (McNulty et al. 2007). Although the role of NPWT in bone healing remains unclear, several basic studies have confirmed that it promotes the differentiation of mesenchymal cells into an osteogenic phenotype and induction of osteoblast differentiation in vitro and promotes bone regeneration in vivo (Zhu et al. 2014; Liu et al. 2018b; Zhang et al. 2022; Yang et al. 2014; Wang et al. 2020). The group of Zhu et al. (2014) and Liu et al. (2018b) investigated

the association between NPWT and osteogenesis using rat mesenchymal cells. Rat periosteumderived mesenchymal stem cells (P-MSCs) and muscle-derived stem cells (MDSCs) were used to envisage closed fractures and open fractures with periosteal avulsion, respectively. Both cells were cultured under continuous negative pressure of -125 mmHg for 3 days, and cell proliferation was found to be significantly enhanced compared to that in culture under atmospheric

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pressure (Zhu et al. 2014; Liu et al. 2018b). Alkaline phosphatase (ALP) activity and mineralization were enhanced in P-MSCs, and increased expression of collagen type 1 and integrin β5, a mechanotransduction-related factor, was observed (Zhu et al. 2014). Experiments using MDSCs and inhibitors suggested that NPWT may promote osteogenic differentiation via the MAPK pathway (Liu et al. 2018b). It was also reported that NPWT induced MSC osteoblast differentiation in vitro and promoted bone regeneration in a rat model of cranial defect in vivo (Zhang et al. 2022). Yang et al. (2014) showed that intermittent negative-pressure exposure predominantly increased ALP activity and type I collagen and VEGF expression in MSCs as well as promoted and induced differentiation into osteoblasts. Wang et al. (2020) showed that 12 h of -125-mmHg negative-pressure exposure significantly increased osteoblast proliferation and ALP activity and promoted maturation.

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of myelinated nerve fibers than did the conventional group. Wu et al. (2018) also treated a rabbit sciatic nerve injury model for 12 weeks in three groups (0 kPa, -20 kPa, and -40 kPa) and reported that electrical nerve conduction velocity, myelinated nerve fiber count, and S100 and BDNF expression were significantly increased in the -20 kPa group compared with those in the 0 kPa group, similar to the findings of Hu et al. Conversely, the -40 kPa group had significantly lower electrical nerve conduction velocity and number of myelinated nerve fibers than did the 0 kPa group, and S100 and BDNF expression levels were not significantly different between -40 kPa and 0 kPa groups. Based on these results, they pointed out that NPWT may be beneficial for nerve repair when used at the appropriate strength but may not contribute to nerve repair when used at too high an intensity (Wu et al. 2018).

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High bacterial loads inhibit wound healing; the effect of normal NPWT on bacterial load is debatable (Patmo et al. 2014). Morykwas et al. (1997) showed in their porcine experiments that negative-pressure treatment at -125 mmHg for 4 days significantly reduced bacterial counts. Ngo et al. (2012) reported a significant reduction of Pseudomonas aeruginosa biofilm in in vitro experiments using agar. Wang et al. (2016) further found that negative-pressure treatment significantly suppressed the secretion of virulence factors, such as exotoxin A, rhamnolipid, and elastase by P. aeruginosa as well as the expression levels of these regulatory genes. Conversely, Boone et al. (2010), in their experiments with pigs, reported that bacterial counts continued to increase after 7 days of NPWT despite gross and microscopic wound improvement. Fujiwara et al. (2013), in their in vitro study, reported that NPWT increased the growth potential of Escherichia coli, with this trend being stronger with intermittent negative pressure than with continuous negative pressure and with short

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The knowledge of the effects of NPWT on peripheral nerve regeneration is limited. A previous study indicated that NPWT for full-thickness skin wounds in diabetic mice significantly increased nerve fiber density and expression of substance P, calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF) in the epidermis and dermis (Younan et al. 2010). The authors stated that NPWT may help in the healing of denervated wounds, such as pressure ulcers and diabetic foot lesions, by modulating the production of nerve fibers and neuropeptides. The effects of NPWT on thicker nerve repair were studied using a rabbit sciatic nerve model. Hu et al. (2015) transected the sciatic nerve of a rabbit by 1 cm, sutured it in its original position, and treated it with NPWT and conventional methods. At 4 and 8 weeks post-operation, the NPWT group had significantly higher electrical nerve conduction velocity, higher intensity of immunohistochemical staining for brain-derived neurotrophic factor (BDNF), and higher number

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cycles than with long cycles. In a prospective randomized trial using tissue bacterial culture specimens from patients with full-thickness wounds that could not be temporarily closed due to wound healing inhibitors, Mouës et al. (2004) reported that the -125-mmHg negative-pressure treatment group showed a significant decrease in gram-negative bacillus counts and a significant increase in Staphylococcus aureus counts compared to those in the conventional treatment group. Assadian et al. (2010) reported no difference in S. aureus abundance with or without negative pressure in an in vitro study with pork, noting that results may differ for live tissues. They mentioned that NPWT-induced changes in bacterial load may not be due to pure negative pressure alone. Biermann et al. (2019) described that the different responses among bacterial species to hypoxia in the dressing may be related to bioburden changes. Based on these considerations, the application of NPWT on wounds with strong infection is generally considered a relative contraindication (Normandin et al. 2021). Efforts have been made to apply NPWT to difficult-to-heal wounds caused by infection, including using antimicrobial materials (Payne and Ambrosio 2009) and liquid infusion in combination with wound cleansing (Wolvos 2004). Silver-impregnated foam is often used as an antimicrobial material. It is assumed that silver ions released slowly into the exudate exert an antimicrobial effect. Most of the ions are recovered without deposition on the wound surface or distribution throughout the body (Abarca-Buis et al. 2014). In vitro studies have shown the antimicrobial efficacy of silver-impregnated foam against S. aureus colonies (Valente et al. 2016; Matiasek et al. 2017) and biofilms, such as those of methicillin-resistant S. aureus (MRSA) (Valente et al. 2016; Ellenrieder et al. 2015) and P. aeruginosa (Valente et al. 2016; Ngo et al. 2012). In clinical practice, its usefulness in acute lower extremity trauma is also described (Hahn et al. 2019). NPWT with irrigation includes (1) irrigation and application of negative pressure continuously and simultaneously (Kiyokawa et al. 2007) and (2) periodically and sequentially

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instilling washing solution, immersion, and negative-pressure treatment (Wolvos 2013; Jerome 2007; Kim et al. 2014); the latter approach is more popular. The infusion solution includes saline solution and disinfectants, such as sodium hypochlorite solution (Yang et al. 2017), povidone-iodine solution (Tahir et al. 2018), and polyhexanide (Timmers et al. 2009). These disinfectants are all considered more effective in wound healing than NPWT without irrigation (Tahir et al. 2018), but some reports suggest that patient outcomes are no different with antiseptic and saline (Kim et al. 2015; Lavery et al. 2020). It has been suggested that the effect cannot be explained by a reduction in bacterial count alone (Burusapat and Sringkarawat 2021), and further study is needed on the optimal composition of the infusion solution and treatment protocol (Saeg et al. 2021).

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Effects on Tumorigenesis

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As mentioned previously, since NPWT promotes cell proliferation and angiogenesis, applying NPWT to sites where malignant tumors are present is contraindicated as it may increase tumor cell growth (Normandin et al. 2021). However, owing to ethical concerns, there is little evidence of whether NPWT leads to proliferation of tumor cells in clinical practice; this aspect has also not been explored in vitro. Continuous or intermittent mechanical stimulation in breast cancer cells enhances invasiveness by promoting EMT (Liu et al. 2018a; Tse et al. 2012) (Fig. 4). In clinical practice, attempts have been made to use NPWT as coverage for wounds after resecting epithelial or mesenchymal malignancies until two-stage reconstruction, assuming complete resection of tumor cells (Fourman et al. 2022; Dadras et al. 2022). Several recent reports have documented that NPWT after resection of malignant tumors may predominantly reduce postoperative complications, such as infection, with no clear difference in the risk of local recurrence of tumors compared to that observed with conventional dressing (Hays et al. 2022; Wang et al. 2022).

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As described previously, NPWT has traditionally been used for acute and chronic open wounds. In recent years, the effect of negative pressure on areas where no raw surface exists has been investigated. NPWT, particularly for primary closed wounds, called incisional NPWT (iNPWT), has attracted increasing attention. iNPWT is performed, instead of regular dressings, on primary closed incisional wounds and is thought to reduce wound complications by using negative-pressure loading, such as with sNPWT devices (Scalise et al. 2016). In the 2022 Cochrane review, it was noted that while iNPWT reduces the incidence of surgical site infection (SSI), wound dehiscence is similar to that observed with standard dressings and may increase blistering problems (Norman et al. 2022). There is no consensus regarding the effect of iNPWT on scar formation (Zwanenburg et al. 2021; Timmermans et al. 2022). These effects of iNPWT may result from tissue or bacterial responses to the primary mechanisms and secondary effects described so far, but further studies are needed to elucidate the detailed mechanisms (Scalise et al. 2016). The efficacy of NPWT in skin graft surgery was first reported in 1998 by Blackburn et al. (1998). Many subsequent clinical studies have shown that fixing grafted skin with NPWT improves the rate of graft skin takes, compared to conventional skin fixation methods (Petkar et al. 2011; Yin et al. 2018). From a macroscopic perspective, several mechanisms have been considered, including suppression of seroma and hematoma development, which can cause poor graft implantation, and good fixation to the graft bed by compression (Yin et al. 2018). Although the molecular biological mechanism is currently unknown, ex vivo studies using patients’ excess skin showed that NPWT promoted the expression of fibroblast growth factor 1 (FGFR1), endothelin 2 (EDN2), and 22 keratinrelated proteins and genes in the graft (Rapp et al. 2020). Animal studies have demonstrated that NPWT enhances the survival area of random pattern flaps (Morykwas et al. 1997). Some researchers believe that NPWT in flap surgery has benefits beyond

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Effects on Operative Wounds

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the reduction in SSI obtained with iNPWT. In the literature comparing postoperative coverage of free myocutaneous flaps with NPWT and conventional treatment using biopsy specimens of the flap tissue, tissue infiltration of CD68-positive macrophages and expression of the inflammatory cytokines IL-1β and TNF-α were reduced in the NPWT group, and the number of apoptotic cells was also reduced due to improved microcirculation (Eisenhardt et al. 2012). Some clinical reports suggest that the drainage effect of NPWT is useful in reducing flap congestion, one of the major causes of flap failure (Yu et al. 2017; Boissiere et al. 2021). Overall, postoperative NPWT coverage after skin flap surgery may have beneficial effects, such as improving skin flap viability and reducing congestion through anti-inflammatory effects and improved microcirculation, but additional research is needed. There have been other attempts at flap preconditioning (Rhodius et al. 2018), in which negative pressure is applied in advance to the area where the flap is to be harvested to obtain improved viability of the flap through the angiogenesis effect, and there is potential for further development (Rhodius et al. 2018; Aydin et al. 2019; Hong et al. 2019; Mohan et al. 2020; Brown and Ghareeb 2021).

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Drawbacks of NPWT

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As described thus far, NPWT works favorably for a variety of wounds through four main mechanisms and accompanying secondary effects; however, there are several drawbacks, including complications and conditions that require caution. We briefly introduce them in this section.

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Complications

Several complications of NPWT have been reported, ranging from minor to serious ones, but most are due to human factors, such as poor patient selection or procedural problems, and almost all complications can be prevented or avoided (Normandin et al. 2021).

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Heart rupture, one of the fatal NPWT-related complications, may be caused by contact between the right ventricle and the edge of incised sternum due to the change in wound geometry (Sartipy et al. 2006; Vos et al. 2013). Preventive measures include covering the heart and immobilizing the sternum (Vos et al. 2013); the use of protective devices inserted between the heart and sternum has also been reported to be effective (Lindstedt et al. 2011; Ingemansson et al. 2014). Direct application of NPWT to exposed internal organs or blood vessels can cause organ damage, such as intestinal fistulas or major bleedings (White et al. 2005; Rao et al. 2007; Fischer 2008). Attention should be paid to vital structures by protecting organs with contact layers or covering them with grafts or flaps (Huang et al. 2014; Normandin et al. 2021). As noted above, infection is one of the most well-known complications of NPWT, although there is ongoing debate. Clinically, the foam itself is a foreign material, and blood clots from bleeding can cause bacterial growth; therefore, treatment should be interrupted if bleeding is suspected (Li and Yu 2014). Pain is another complication that should not be ignored. Pain in NPWT occurs at the start of treatment and during dressing changes. Pain during treatment has been reported to be stronger with intermittent negative pressure than with continuous negative pressure (Borgquist et al. 2010a; Malmsjo et al. 2012); starting treatment at low pressure and gradually increasing the pressure are considered effective (Normandin et al. 2021). There have been reports of the use of anesthetics during dressing changes (Tank et al. 2021); however, large studies are needed to draw any conclusions.

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There are several conditions under which NPWT application requires caution; incorrect patient selection can not only prevent the expected therapeutic effect but also cause the aforementioned complications.

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Contraindications

NPWT for wounds with malignant tumors or exposed vital organs is contraindicated owing to concerns regarding tumor growth and fatal complications, as mentioned above, and is considered only after thorough resection of tumor cells and organ protection (Normandin et al. 2021). It is important to consider the possibility of rather harmful effects on tissue blood flow, such as when applying NPWT to ischemic lesions or when using NPWT in the extremity circumferentially (Kairinos et al. 2009a, b; Livingstone et al. 2021; Singh et al. 2021). If NPWT is to be performed on ischemic tissue, it should only be done after appropriate revascularization (De Caridi et al. 2016). When NPWT is performed on wounds with infected or necrotic tissue, such as diabetic foot lesions, proper debridement and control of infection are necessary beforehand, as their persistence may lead to serious systemic complications (Meloni et al. 2015; Chen et al. 2021). With the advent of NPWTi-d, minor colonization can now be treated relatively safely (Kim et al. 2014).

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Final Remarks

A quarter of a century has passed since the advent of NPWT, and it is time for basic research to support its effectiveness. NPWT has many variable parameters, such as the type of treatment device, dressing material, intensity and duration of negative pressure, duration of treatment, continuous or variable pressure, presence or absence of irrigation, composition of irrigation solution, and even patient-side factors, which must be fully considered when designing the study model. Moreover, when conducting in vivo experiments in mice, it is difficult to confirm the effects of the experimental model on the physiological functions of the whole body of the mouse and the possible adverse effects on the experimental results (Dastouri et al. 2011). Furthermore, different devices may have different mechanisms and effects (Nuutila et al. 2021), and it is important to consider whether the findings of each report apply

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Negative-Pressure Wound Therapy: What We Know and What We Need to Know

to NPWT, as a whole. We believe that elucidation 1023 of the mechanism of NPWT through expansion 1024 of basic research will greatly contribute to the 1025 expansion of therapeutic indications, such as 1026 safer use with reduced complications, in addition 1027 to the improvement of devices or combination 1028 with other therapeutic methods with newly 1029 defined therapeutic targets. 1022

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Acknowledgments This study was partially supported by JSPS KAKENHI Grant Number 20 K12725. We would also like to thank Editage (www.editage.com) for English language editing.

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Conflict of Interest The authors declare no conflict of interest.

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Ethical Approval Ethical approval is not applicable, as this article does not present any original unpublished research conducted in human participants or animals.

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Yang Z, Yao JF, Xu P, Zhang JB, Zhang YM, Zhu YJ et al (2014) Functions and mechanisms of intermittent negative pressure for osteogenesis in human bone marrow mesenchymal stem cells. Mol Med Rep 9:1331–1336. https://doi.org/10.3892/mmr.2014.1968 Yang C, Goss SG, Alcantara S, Schultz G, Lantis Ii JC (2017) Effect of negative pressure wound therapy with instillation on bioburden in chronically infected wounds. Wounds 29:240–246 Yin Y, Zhang R, Li S, Guo J, Hou Z, Zhang Y (2018) Negative-pressure therapy versus conventional therapy on split-thickness skin graft: a systematic review and meta-analysis. Int J Surg 50:43–48. https://doi.org/10. 1016/j.ijsu.2017.12.020 Younan G, Ogawa R, Ramirez M, Helm D, Dastouri P, Orgill DP (2010) Analysis of nerve and neuropeptide patterns in vacuum-assisted closure-treated diabetic murine wounds. Plast Reconstr Surg 126:87–96. https://doi.org/10.1097/PRS.0b013e3181da86d0 Younan GJ, Heit YI, Dastouri P, Kekhia H, Xing W, Gurish MF et al (2011) Mast cells are required in the proliferation and remodeling phases of microdeformational wound therapy. Plast Reconstr Surg 128:649e–658e. https://doi. org/10.1097/PRS.0b013e318230c55d

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Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 153–166 https://doi.org/10.1007/5584_2023_774 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 Published online: 31 May 2023

Vitamin A Deficiency, COVID-19, and Rhino-Orbital Mucormycosis (Black Fungus): An Analytical Perspective Aziz Rodan Sarohan , Sait Edipsoy , Zeynep Gürsel Özkurt , Can Özlü , Ayça Nur Demir and Osman Cen Abstract

Mucormycosis is a rare but serious opportunistic fungal disease characterized by rhinoorbito-cerebral and pulmonary involvement. It is mainly seen in people with secondary

A. R. Sarohan (✉) Shagreen Health Life Science, İstanbul, Turkey e-mail: [email protected] S. Edipsoy Department of Ophthalmology, Medicina Plus Medical Center, İstanbul, Turkey e-mail: [email protected] Z. G. Özkurt Princess Margaret Cancer Center Ocular Oncology Clinic, Toronto, ON, Canada e-mail: [email protected] C. Özlü Department of Hematology, Kütahya Health Science University, Evliya Çelebi Education and Research Hospital, Kütahya, Turkey e-mail: [email protected] A. N. Demir Faculty of Medicine, Afyonkarahisar Health Science University, Afyon, Turkey e-mail: [email protected] O. Cen Feinberg School of Medicine, Northwestern University, Chicago, IL, USA Waubonsee College, Sugar Grove, IL, USA e-mail: [email protected]

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immunosuppression, isolated vitamin A deficiency, measles, and AIDS patients. It showed a rise during the second wave of the COVID19 epidemic in the spring of 2021 in India, especially in diabetic COVID-19 patients. Vitamin A deficiency is known to cause nutritional immunodeficiency and hence leading the way to increased opportunistic fungal, bacterial, and viral infections. In the eye, it causes keratitis, night blindness, xerophthalmia, conjunctivitis, Bitot spots, keratomalacia, and retinopathy. It also causes decreased tear secretion and deterioration of the anatomical/ physiological defense barrier of the eye. The negative impact of vitamin A deficiency has been previously demonstrated in measles, AIDS, and COVID-19. We think that mucormycosis in COVID-19 might be rendered by vitamin A deficiency and that vitamin A supplementation may have preventive and therapeutic values against mucormycosis and other ocular symptoms associated with COVID-19. However, any vitamin A treatment regimen needs to be based on laboratory and clinical data and supervised by medical professionals. Keywords

Black fungus · Conjunctivitis · COVID-19 · Keratitis · Mucormycosis · Night blindness · Vitamin A deficiency · Xerophthalmia 153

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Introduction

The number of life-threatening fungal infections has increased dramatically over the past two decades. Recent estimates have identified a global disease burden of approximately two million patients with systemic and invasive fungal infections (Bongomin et al. 2017; Brown et al. 2012; Kim 2016). However, despite aggressive treatments, opportunistic fungal infections in high-risk patients have an unacceptably high mortality rate (Bongomin et al. 2017; Brown et al. 2012). The main reason for this is the rapid increase in the number of chronically immunocompromised patients (Brown et al. 2012; Mei-Sheng Riley 2021). Aggressive chemotherapy applications to treat leukemia and other hematological malignancies, the increase in bone marrow transplantations, and the widespread use of immunosuppressive drugs to prevent rejection of transplanted organs, as well as the persistence of AIDS as a global epidemic, have significantly increased the number of immunosuppressed patients who are susceptible to the opportunistic infections including mucormycosis (Brown et al. 2012; Mei-Sheng Riley 2021; Reid et al. 2020). Mucormycosis, also known as zygomycosis or inappropriately black fungus, is a rare but serious opportunistic fungal disease characterized by rhino-orbito-cerebral, pulmonary, systemic, gastrointestinal, and cutaneous involvement (Binder et al. 2014; Petrikkos et al. 2012; Reid et al. 2020; WHO 2021). Mucorales spores are common in various environments and nasal flora of many people and normally do not cause invasive clinical pictures in immunocompetent individuals but can cause severe and fatal mucormycosis in the immunosuppressed individuals by invading the nasal cavities, paranasal sinuses, and from there to the orbit and the brain (Gandra et al. 2021; Jeong et al. 2019). As an opportunistic infection, mucormycosis is mainly seen in immunosuppressed individuals, such as those with AIDS, diabetes, and chemotherapy treatment for malignancies (Binder et al. 2014; Jeong et al. 2019; Reid et al. 2020). It is also seen in isolated

vitamin A deficiency, non-diabetic COVID-19 patients, and measles (Dwivedi et al. 2023; Lagorce Pagès et al. 2000; Mei-Sheng Riley 2021). The second wave of the COVID-19 pandemic had shaken India, reaching more than 400,000 daily COVID cases in the spring of 2021 leading to its healthcare system being on the verge of collapse. In this second wave, mucormycosis was observed at an alarming level between April and June of 2021, which had further shaken the country’s healthcare system (Bhattacharyya et al. 2021a, b; Dilek et al. 2021; Lima et al. 2021b; Nagalli and Kikkeri 2021; Sen et al. 2021). These cases of COVID-19-associated mucormycosis (CAM) increased simultaneously in multiple states, causing a second outbreak within the COVID-19 pandemic and constituting about 71% of global CAM cases (Gambhir et al. 2021). This sudden increase in mucormycosis in India was devastating as it led to orbital exenteration in some patients. With the spread of ocular infarction, some patients’ eyes need to be surgically removed, to prevent the dissemination of the infection into the brain (Gandra et al. 2021). Although the cause of this sharp rise of mucormycosis in the COVID-19 epidemic is still unknown, its frequent occurrence in diabetic COVID-19 patients may indicate its possible association with diabetes (Al-Tawfiq et al. 2021; Bhattacharyya et al. 2021b; Dilek et al. 2021; Dwivedi et al. 2023). Diabetes, overuse of steroidal therapeutics, and tocilizumab have been attributed to the mucormycosis spike (Dwivedi et al. 2023; Gandra et al. 2021; Khichar et al. 2021). Diabetes is known to weaken the immune system and makes the individual susceptible to other diseases/infections (Rocha et al. 2021). It is also noted that the widespread use of antihyperglycemic and immunosuppressive drugs might weaken the immune system and lead to opportunistic fungal infections (Elhamamsy et al. 2021; Rocha et al. 2021). We hypothesize that hypovitaminosis A-associated immune deficiency is most likely a major underlying cause of the increased mucormycosis cases during the second wave of the COVID-19

Vitamin A Deficiency, COVID-19, and Rhino-Orbital Mucormycosis (Black Fungus). . .

pandemic in India. A possible depletion of vitamin A in culminating COVID-19 infection may compromise a proper immune response leading to secondary infections as seen in measles and HIV infections (Bello et al. 2016; Mselle 1999; Rodrigues and Dohlman 2004). In this study, we aim to bring attention to the possible role of vitamin A in COVID-19 and associated infections, including mucormycosis. We discuss the relationship between vitamin A deficiency and COVID-19, measles, AIDS, and other chronic diseases. In particular, the proven efficacy of vitamin A in reducing measles mortality and related eye complications is reviewed. In this context, we believe that vitamin A or retinoic acid supplementation may prove therapeutically beneficial in treating and preventing mucormycosis and other ocular infections associated with COVID-19 especially early during infection. We think this idea deserves a well-controlled comprehensive study. However, it should be noted that the therapeutic use of vitamin A has to be managed by medical professionals.

2

Mucormycosis, COVID-19, and Vitamin A Deficiency

COVID-19 infection displays a plethora of multiorgan involvement including pulmonary, cardiac, circulation, immune, and ocular involvement (Gavriatopoulou et al. 2020; Lanska 2010; Munjal et al. 2020; Romero-Sánchez et al. 2020). The common ophthalmic manifestations include central retinal artery occlusion, central retinal vein occlusion, cranial nerve palsy, follicular conjunctivitis, optic neuritis, conjunctivitis, and mucormycosis (Sen et al. 2021; Wan et al. 2022). Ocular mucormycosis is characterized by swelling and pain in the eyes, dark spots around the nose, and an excessively runny nose. Blurred vision, loss of vision, and eyelid loss have also been reported in some patients (Gandra et al. 2021). Ocular findings have been observed as late symptoms of the disease in COVID-19 and mucormycosis tends to occur approximately 2 weeks after recovery, leading to

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rehospitalization (Lima et al. 2021b; Sen et al. 2021; Wan et al. 2022). Vitamin A has an important role in development, tissue repair, vision, and immunity (Ross and Stephensen 1996; Sommer 2008; Sommer and Vyas 2012; Tanumihardjo 2011). In the eye, it is essential in the biochemistry of the vision (Faustino et al. 2016). Its deficiency abrogates the biochemistry of the vision and leads to the development of microbial keratitis, night blindness, xerophthalmia, conjunctivitis, Bitot spots, keratomalacia, and retinopathy (Sherwin et al. 2012; Sommer 1983, 1993). Vitamin A deficiency is classically caused by food deprivation and is common in underdeveloped countries; rural areas and peripheries of large cities in South Asia, Africa, and Latin America; and poor communities of large cities in developed countries (Mason et al. 2015; Melo et al. 2004; Spannaus-Martin et al. 1997). It is a global health problem and has been the target of international preventive vitamin A supplements and periodic evaluation programs (Akhtar et al. 2013; Mason et al. 2015). The most vulnerable people are children, pregnant women, and the elderly. The prevalence of hypovitaminosis A in children under 6 years of age reaches 50% in some regions (Akhtar et al. 2013). In addition to ocular problems, hypovitaminosis A causes growth retardation, infertility, congenital malformations, susceptibility to infections, and premature death (Akhtar et al. 2013; Mason et al. 2015). The risk of developing ocular symptoms in COVID-19 has been linked to high viral load (Wan et al. 2022). About one-fifth (21%) of the cohort of patients diagnosed with COVID-19 reported one or more symptoms related to dry eye disease, of which the most frequent ones were turbidity (9.2%), itching (6.1%), and pain or burning sensation (4.8%) (Wan et al. 2022). The clinical “pink eye” picture commonly seen in severe COVID-19 patients is similar to conjunctivitis observed in vitamin A deficiency (Al-Namaeh 2021; Sen et al. 2021). We believe that the cause of the eye findings in COVID-19 might be due to vitamin A deficiency (Sarohan 2020). We previously hypothesized that COVID19 infection might cause depletion of vitamin A

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in the body leading to immunosuppression, biphasic immune dysregulation, defect in type-I interferon synthesis, cytokine storm, excessive inflammatory response, systemic organ damage, and aggravated clinical picture (Sarohan 2021b; Sarohan et al. 2021). In a recent study, we showed that vitamin A levels were low in severe COVID19 patients (Sarohan et al. 2022). It will be important to elucidate if COVID-19 patients develop retinitis and other vision problems due to atrophy and necrosis of cells in the cornea and or retina resulting from vitamin A deficiency and consecutively disturbed retinoic acid signaling.

2.1

Mucormycosis Superinfection in COVID-19

Most CAM cases in India in 2021 were observed in diabetic COVID-19 patients (Banerjee et al. 2021; Bhattacharyya et al. 2021a; Hoenigl et al. 2022; Nagalli and Kikkeri 2021). The widespread use of corticosteroids in COVID-19 treatment to suppress the excessive inflammatory response and cytokine storm might have further predisposed diabetic patients to the fungal disease (Bhogireddy et al. 2021; Hoenigl et al. 2022; Patel et al. 2021). However, it is important to note that CAM is also seen in COVID-19 patients who do not have diabetes or have recently used corticosteroids (Nair et al. 2021). In this study, 13 otherwise immunocompetent COVID-19 patients with no previous diabetes nor a history of corticosteroid use were reported to develop a new onset of uncontrolled diabetes and rhinoorbital mucormycosis following COVID-19 infection, and some of these patients underwent orbital exenteration due to advanced orbital involvement despite maximal medical therapy (Nair et al. 2021). Some cases of rhino-orbital mucormycosis requiring aggressive surgery and medical intervention have also presented with a new onset of diabetes following COVID-19 infection (Nair et al. 2021). Age, rhino-orbitalcerebral involvement, and intensive care unit admission have been associated with increased mortality (Nair et al. 2021). In another study, among 287 mucormycosis patients, 187 (65.2%)

had CAM (Patel et al. 2021). Uncontrolled diabetes was the most common underlying condition among CAM and non-CAM patients. In 32.6% of CAM patients, the only underlying disease was COVID-19. Hypoxemia associated with COVID19 and inappropriate use of glucocorticoids have been reported to be independently associated with CAM (Patel et al. 2021). The case death rate due to mucormycosis was 45.7%, which was similar for CAM and non-CAM patients. In addition to India, CAM cases have been reported in other countries such as the USA, the Netherlands, Brazil, England, Germany, Italy, and others (Hoenigl et al. 2022). Four cases of CAM were also reported in the Netherlands between December 2020 and May 2021 (Buil et al. 2021). Mucorales species have been identified as the causative agent of mucormycosis in these patients. Clinical presentations included pulmonary, rhino-orbital, cerebral, and disseminated infection. Three of them have died. All were men over 50 years of age (Buil et al. 2021). In another study, three non-diabetic cases of invasive mucormycosis with different outcomes were presented (Elhamamsy et al. 2021). In this study, the increase in CAM was attributed to both the immune system dysregulation in COVID-19 and the use of high-dose corticosteroids (Elhamamsy et al. 2021). In addition, the burden on the health system with the epidemic, the intense use of oxygen in intensive care units, and the use of non-sterilized water to humidify oxygen have been shown to be a factor in the transmission of mucormycosis (Elhamamsy et al. 2021).

2.2

Depletion of Vitamin A in COVID-19 Infection

Vitamin A depletion is a common pathogenetic mechanism observed during some infections, such as measles and HIV (WHO 2017). Depletion of vitamin A during infections may lead to systemic organ involvement associated with persistent infections, reinfections, secondary infections, and increased inflammation (Sarohan et al. 2021). A few studies from Brazil and China show a high rate of vitamin A deficiency, especially in

Vitamin A Deficiency, COVID-19, and Rhino-Orbital Mucormycosis (Black Fungus). . .

rural and urban areas (Chen et al. 2017; Custodio et al. 2009; Lima et al. 2021a; Nascimento et al. 2007). A 2009 screening study found that 17.4% of children and 12.3% of women were deficient in vitamin A throughout Brazil with high prevalence in rural and urban areas (Custodio et al. 2009). In another similar study, vitamin A deficiency was found in 26% of the elderly population aged over 60 years living in big cities (Nascimento et al. 2007). These data show a strong correlation between the epidemiology of vitamin A and the geographical and demographic course of COVID-19 and may suggest that vitamin A deficiency may be an underlying reason for the concentrated COVID-19 infection in urban areas and the elderly population. It is also well known that COVID-19 has a severe clinical course and high mortality in urban areas and especially in the elderly population (Nascimento et al. 2007). However, unfortunately, no epidemiological study has been conducted to prove this relationship. In yet another study conducted in Brazil, the high risk of death from COVID-19 in the southeastern part of the country, where the major cities are located, was attributed to the high proportion of the elderly (Lima et al. 2021a). The same study also noted the high rates of infection and mortality for COVID-19 among young adults, people of low socioeconomic status, and people without access to healthcare in the less developed parts of the north of the country (Lima et al. 2021a). The relationship between COVID-19 and advanced age was especially emphasized in the study (Lima et al. 2021a). The result of this study indirectly points to a possible relationship between vitamin A deficiency and COVID-19. However, the study did not examine the relationship of COVID-19 with micronutrient deficiencies such as vitamins A, D, or others. The prevalence of vitamin A deficiency in the elderly population over 60 years old living in big cities was found to be as high as 22% in China and 26% in Brazil (Chen et al. 2017; Lata et al. 2021).

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Role of Vitamin A in Immunity and Inflammation

Vitamin A has paramount importance in development, tissue repair, vision, and immunity. In immunity, vitamin A is recognized as an important factor for the development of a proper immune response, suppression of acute inflammation, and tissue repair after disease or injury (Green and Mellanby 1928; Stephensen and Lietz 2021; Timoneda et al. 2018). Vitamin A and its derivative retinoic acids have anti-infective, anti-inflammatory, and strong adjuvant activities (Hao et al. 2021; Li et al. 2020; Midha et al. 2020). Retinoic acid signaling is important in the synthesis of type-I interferon, which is a critical antiviral mediator in strengthening the immune system and clearing viruses (Kell and Gale Jr. 2015; Liu et al. 2016). In vitamin A deficiency, retinoic acid is depleted, type-I interferon synthesis is disrupted, and host defense is weakened (Sarohan et al. 2021). The decrease and/or depletion of vitamin A in the body disrupts type-I interferon synthesis and weakened immune response leading to an increased susceptibility to infections (Sarohan 2020; Sarohan et al. 2021). Therefore, vitamin A and retinoic acids are vital for an effective immune defense during viral infections. Vitamin A deficiency displays an acquired immunodeficiency-like picture (Oliveira et al. 2018; Stephensen 2001; Timoneda et al. 2018), which leads to increased opportunistic fungal, bacterial, and viral infections (Ross and Stephensen 1996; Stephensen 2001; Tanumihardjo 2011). While the earliest signs of vitamin A deficiency manifest themselves in immune cells (Hester et al. 2020; Timoneda et al. 2018), the eye-related signs are manifested later (Gilbert 2013; Sommer 2008). Vitamin A deficiency-related immune signs are decreased interferon response to viral infections and excessive chronic inflammation and cytokine storm leading to multiorgan damage, such as liver, lung, and kidney (Sarohan 2020; Sarohan et al. 2021).

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Vitamin D has gained more attention than vitamin A in the treatment of COVID-19 infection (Abdulateef et al. 2021; Vyas et al. 2021). Both vitamin A and vitamin D are lipid in structure and relay their signaling through retinoid receptors. In a systematic review, the authors listed a number of candidate molecules, including vitamin A, vitamin D, and zinc, as essential components for the development of an adequate immune response against COVID-19 infection (Zhang and Liu 2020). Zinc is also shown to modulate vitamin A metabolism and immune system regulation in infections including fungal pathogenicity (Staats et al. 2013). The protective effect of vitamins A and D against COVID-19 supports our initial hypothesis in retinol depletion and retinoid signaling disorder that we initially proposed to explain the pathogenesis of COVID19 (Sarohan et al. 2021). Vitamin A deficiency is associated with a variety of infectious diseases, including diarrhea, respiratory diseases, measles, and HIV-1 infection (Bello et al. 2016; Stephensen 2001; Stephensen and Lietz 2021). Some infections, such as measles and HIV, may lower the systemic vitamin A levels and consequently disturbed retinoid signaling (Arrieta et al. 1992; D’Souza and D’Souza 2002b) that leads to systemic inflammation and severe clinical pictures (Stephensen 2001; Timoneda et al. 2018). This reciprocal relationship between vitamin A and infections in the host-pathogen interaction causes a vicious cycle during infections, increasing infection-induced morbidity and mortality (Timoneda et al. 2018; Wiseman et al. 2017). Similar studies point to the importance of vitamin A in the COVID-19 pathogenesis as well. More than 71% of COVID19 patients had a low level of vitamin A, and decreased level of vitamin A was associated with the severity of COVID-19 infection (Tepasse et al. 2021; Tomasa-Irriguible et al. 2021). Furthermore, an in silico analysis suggested that vitamin A might be a candidate for the treatment of COVID-19 infection (Chakraborty et al. 2022). Many trials have been conducted with vitamin A to treat and prevent various infections (Green et al. 1931; Sommer 2008; Sommer et al. 1983).

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In one of the most important studies, administering daily vitamin A reduced mortality rates by 50% in measles cases admitted to Grove Fever Hospital outside London (Ellison 1932). Sulfa-based antimicrobials introduced before the Second World War and some other antibiotics discovered afterward were found to be much more effective than vitamin A in the treatment of acute bacterial infections. With the increase in the welfare level of societies and the improvement in their nutritional status, vitamin A deficiency and its clinical findings (especially xerophthalmia) have almost completely disappeared. Afterward, clinical studies and reports on vitamin A have not attracted much attention (Sommer 2008). After these developments, research on the administration (and advocacy) of vitamin A to treat and prevent infections has also come to almost a standstill leading to a loss of interest in studies on vitamin A deficiency (Sommer 2008; Sommer and Vyas 2012). However, the epigenetic role and mechanisms of vitamin A in regulating gene functions remain a subject of exploration and interest (Sommer 2008). The association of chronic inflammatory diseases such as diabetes, autoimmune diseases, and chronic degenerative neurological diseases such as epidemics with malnutrition and vitamin A deficiency will revive the interest in vitamin A. In addition, the increased antibiotic resistance shifts the attention to alternative approaches in treating bacterial infections and boosting a proper immune response with vitamins being such a prominent approach (Mora et al. 2008).

2.4

Role of Vitamin A in Eye Health

Vitamin A is extremely important in the biochemistry of vision and the health of the eye (Faustino et al. 2016; Lanska 2010; Sommer 1983). Its deficiency leads to the development of night blindness, xerophthalmia, conjunctivitis, Bitot spots, microbial keratitis, corneal ulceration, keratomalacia, and retinopathy (Chung et al. 2022; Connell et al. 2006; Morjaria et al. 2011; Sherwin et al. 2012; Sommer 1993). Vitamin A deficiency also causes decreased tear secretion

Vitamin A Deficiency, COVID-19, and Rhino-Orbital Mucormycosis (Black Fungus). . .

and deterioration of the anatomical/physiological defense barrier of the eye leading the way to opportunistic ophthalmic infections (Sherwin et al. 2012; Sirisinha 2015). A central figure in human nutritional research, McLaren designated vitamin A as a representative vitamin for eye and vision (McLaren 2000). Mild xerophthalmic Indonesian children with night blindness and Bitot spots were reported to die at much higher rates than their non-xerophthalmic peers (Akhtar et al. 2013; Sommer et al. 1983). This association between mortality rates and the severity of xerophthalmia suggested that even “subclinical” vitamin A deficiency not accompanied by ocular changes could be associated with increased mortality, which reveals that prevention of vitamin A deficiency can lead to massive reductions in overall mortality rates (Stevens et al. 2015). Night blindness and xerophthalmia are among the first ocular signs of vitamin A deficiency (Faustino et al. 2016; Sommer 1983). While night blindness is developed earlier, xerophthalmia is the most important expression of vitamin A deficiency and is pathognomonic for vitamin A deficiency (Sommer 1990, 2008). More severe forms of vitamin A deficiency, such as corneal xerosis, corneal ulceration, and keratomalacia, tend to occur with prolonged malnutrition. In its more severe forms, vitamin A deficiency causes dryness of the cornea, resulting in damage to the cornea and retina, with serious consequences that can lead to blindness (Awasthi et al. 2013; Lanska 2010; Sommer 1990). An estimated 250,000–500,000 children become blind each year due to vitamin A deficiency, and half of these die within 12 months of losing sight (WHO 2009, 2014). Vitamin A deficiency is associated with significant morbidity and mortality from common childhood infections and is the leading preventable cause of childhood blindness in the world (Imdad et al. 2017; WHO 2014). WHO still advocates for prophylactic vitamin A use for the prevention of night blindness and xerophthalmia in less developed countries (Awasthi et al. 2013; WHO 2009).

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Vitamin A deficiency especially weakens its ability to fight infections. Vitamin A is essential for the differentiation of the mucus-secreting epithelium of the eye and the maintenance of tear secretion. Secondary and opportunistic infections, such as mucormycosis, develop in the eye as the anatomical and immunological defense barriers of the eye becomes impaired (Faustino et al. 2016; Imdad et al. 2017; WHO 2014). Night blindness more often occurs during pregnancy, possibly due to the deepening of pre-existing marginal maternal vitamin A deficiency because of the nutritional demands of pregnancy and intervening infections (Awasthi et al. 2013; Hussaini et al. 1978; Tielsch et al. 2008; Wolf 1978). It has been found that 80% of pregnant women develop vitamin A deficiency after week 34 of gestation (Awasthi et al. 2013; Villar et al. 2003). Vitamin A deficiency is associated with serious maternal and fetal complications of pregnancy such as preeclampsia and preterm birth and is also responsible for increased maternal and fetal death and other poor outcomes of pregnancy and lactation (Awasthi et al. 2013; Villar et al. 2003). One study reported that a 40% reduction in maternal mortality could be achieved with routine supplementation of vitamin A during pregnancy (West et al. 1999). Although eye problems due to vitamin A deficiency are rare in developed countries, they usually present as superposed microbial keratitis. In addition, a wide spectrum of ocular findings can be encountered, ranging from simple night blindness, Bitot spots, and conjunctival and corneal xerosis to serious corneal ulcers, scar formation, and corneal perforation (Chung et al. 2022; WHO 2014). Sometimes, vitamin A deficiency manifests itself with subclinical findings. Interestingly, the patient may have severe vitamin A deficiency, despite the absence of classical ocular findings of hypovitaminosis A (Mason et al. 2015; WHO 2014). Late-occurring ocular findings can develop insidiously and rarely occur before the age of two (Diab and Krebs 2018). Therefore, vitamin A levels begin to decline before the classic ocular manifestations of vitamin A deficiency appear.

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2.5

A. R. Sarohan et al.

Vitamin A Deficiency in Measles and Eye Involvement

COVID-19 infection may mimic measles infection, especially for depleting vitamin A and dysregulating immune response. COVID-19 infection displays many similarities to that of measles. For example, the following findings are similarly observed in both infections: depletion of vitamin A, predisposition to secondary infections, aggravation of the clinical picture, pneumonia, and ARDS (Midha et al. 2020; Sarohan et al. 2022; Tepasse et al. 2021). It has been shown that the severity of measles, a disease characterized by the suppression of the immune system and infectious complications, is directly proportional to the degree of vitamin A deficiency (D’Souza and D’Souza 2002a; Imdad et al. 2017; Sherwin et al. 2012). As in measles, vitamin A deficiency causes the development of secondary infections such as mucormycosis in COVID-19 (Imdad et al. 2017; Sarohan 2020, 2021a; Sommer 1993; Stephensen and Lietz 2021). In an observational clinical study conducted at the beginning of the pandemic, it was found that serum vitamin A levels were low in severe COVID-19 patients (Sarohan et al. 2022). In another study, retinol levels were found to decrease in COVID-19 patients in correlation with the worsening of the clinical picture (Tepasse et al. 2021). Measles is considered a global health threat (WHO 2017). It is a major ongoing problem in developing countries, affecting approximately 30 million children, and causing up to one million deaths annually (Angelo et al. 2019; Crecelius and Burnett 2020). Its prevalence in developed western countries has also risen in recent years (Angelo et al. 2019; Crecelius and Burnett 2020). Especially in the USA and European countries, its prevalence has recently increased (Angelo et al. 2019; Chovatiya and Silverberg 2020). In Ireland, it increased by 244% and became an important public health problem (O’Mahony et al. 2019). Concurrent with the COVID-19 pandemic in New Zealand, cases of measles outbreak-related keratitis occurred in 2020 (Ong et al. 2020). In

measles, conjunctivitis and keratitis are the most common ocular manifestations, while encephalitis and pneumonia may cause mortality (Ong et al. 2020). Measles blindness is the single leading preventable cause of blindness in children in low-income countries and is responsible for an estimated 15,000–60,000 cases of blindness per year (Semba and Bloem 2004). There is a close synergism between measles and vitamin A deficiency, which can result in xerophthalmia, corneal ulceration, keratomalacia, and subsequent corneal scarring and blindness (Crecelius and Burnett 2020; D’Souza and D’Souza 2001; Hussey and Klein 1993; Imdad et al. 2017). High-dose vitamin A supplements are given to all children with measles in developing countries (Bello et al. 2016; Benn 2012). In addition to preventing measles transmission and expanding the scope of measles immunization, interventions to improve children’s vitamin A nutrition and vitamin A prophylaxis programs have gained important momentum among the main strategies to prevent measles blindness (Bello et al. 2016; Sherwin et al. 2012; Sommer 1990). Control of blindness in children has been recognized as a high-priority intervention under the World Health Organization’s VISION 2020, Right to Sight Program (Semba and Bloem 2004; WHO 2007). Measles is destructive to vitamin A metabolism leading to severe vitamin A deficiency in the host during measles infection (Bello et al. 2016; Imdad et al. 2017; Sherwin et al. 2012). During the 2015 measles outbreaks in California, about 50% of the infected children were found to be deficient in vitamin A during the screenings (Arrieta et al. 1992). The World Health Organization added prophylactic vitamin A supplements to their treatment protocols to prevent vision loss caused by vitamin A deficiency in children (Bello et al. 2016; Imdad et al. 2017; Sherwin et al. 2012). Prophylactic vitamin A applications aim to prevent measles-related blindness in countries where measles is common (Bello et al. 2016; D’Souza and D’Souza 2002a). For this purpose, 200,000 IU/day of vitamin A supplementation given to children with measles for

Vitamin A Deficiency, COVID-19, and Rhino-Orbital Mucormycosis (Black Fungus). . .

2 days resulted in a significant reduction in measles-related blindness as well as overall mortality (Bello et al. 2016; Semba and Bloem 2004).

such as vitamin A and vitamin D have been overlooked.

3 2.6

Vitamin A Deficiency in Chronic Diseases and COVID-19

Chronic diseases such as obesity, diabetes, and cardiovascular diseases create comorbidity for COVID-19 and increase COVID-19 mortality by aggravating its clinical picture (Iadecola et al. 2020; Lim et al. 2020; Mertens and Peñalvo 2020; WHO n.d.). In recent years, chronic inflammatory and autoimmune diseases such as obesity, diabetes, cardiovascular diseases, and autism, which are not specifically caused by microorganisms, are associated with an unhealthy lifestyle, nutrition, and vitamin A deficiency (Snelson et al. 2021; WHO n.d.). The uncontrolled increase in chronic inflammatory diseases, which predispose to severe disease in COVID-19 and cause increased mortality and morbidity, especially in western societies, is attributed to unhealthy nutrition, such as high-calorie packaged foods (WHO n.d.). In this “healthy recovery from COVID-19” manifesto, WHO declared that the largest risk factor for mortality and morbidity for COVID-19 is chronic diseases such as obesity, diabetes, and cardiovascular diseases, which are caused by high-calorie unhealthy diet (WHO n.d.). Countries’ vulnerability to the deadly COVID-19 is indicated to be higher with increasing vitamin A deficiency (Mertens and Peñalvo 2020). It has been reported in many recent studies that chronic diseases, which have the potential to harm human health and socioeconomic structure more than COVID-19, are associated with highcalorie malnutrition and vitamin A deficiency (Marley et al. 2021; Nugent 2008; Snelson et al. 2021; WHO 2020). The uncontrolled increase in chronic inflammatory diseases such as obesity and diabetes, which predispose to severe disease in COVID-19 and cause increased mortality and morbidity, especially in western societies, is attributed to unhealthy and high-calorie, packaged foods, whose deficiencies in vital nutrients

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Discussion and Conclusion

Vitamin A was discovered 106 years ago (Semba 2012) and the prevention of vitamin A deficiency at population scales has been recognized as a public health priority for over six decades by the World Health Organization (Daré et al. 2019; Underwood 1994; WHO 2009). It is known that vitamin A supplements strengthen the immune system and protects patients from secondary infections, especially measles and AIDS. For this reason, we believe that vitamin A and retinoic acids can be useful in the prophylaxis and treatment of opportunistic infections and eye problems such as immunosuppression and mucormycosis occurring in association with COVID-19. A controlled randomized clinical trial may produce useful results not only for COVID-19 but also for similar possible future epidemics and pandemics. Despite great combined efforts, the molecular pathogenesis of COVID-19 has not been well identified. In addition, the immune protection induced by the available COVID-19 vaccines wanes very fast within months, and the drugs adopted for the treatment are not very effective (Feikin et al. 2022; Prompetchara et al. 2020; Toor and Chana 2022). The large increase in the population of immunocompromised patients, the limited efficacy of current antifungals, and the increasing resistance to these drugs have prompted a search for alternative solutions for the prevention of invasive mucormycosis (Meir and Osherov 2018). The success of reduced blindness and mortality associated with the prophylactic use of vitamin A in measles outbreaks may serve as a model for the control of COVID19 and COVID-19-related mucormycosis. COVID-19 vaccines, which had been developed at an unprecedented speed, have not provided longlasting effective immunity. Due to the long and costly drug development process, unfortunately, no fully effective drug has yet been found against

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COVID-19. Despite the extraordinary measures, so far more than 650 million people have been infected and more than 6.6 million lost their lives (as of December 2022) (WHO n.d.). In addition, the pandemic has caused very serious socioeconomic losses. The most feared mutations in the pandemic had been our saviors. With this last mutation seen with the Omicron variant, the SARS-CoV-2 virus gained the ability to coexist with the host, as required by the evolutionary process, leading the pandemic toward its end (Fan et al. 2022; Singhal 2022; Tian et al. 2022). However, the virus is evolving, and the rise of dangerous mutants is not totally out of question yet. In addition, it should be kept in mind that the Omicron variant may cause severe infections in elderly and immunocompromised patients and individuals with comorbid diseases. Likewise, even though the COVID-19 pandemic is ending, chronic, autoimmune, and some other degenerative neurological diseases, which are seen after COVID-19 and defined as post-COVID syndromes, and some sequelae caused by vaccines will continue to be a problem for a long time. Therefore, elucidation of the pathogenesis of COVID-19 and associated diseases as well as its modulation of the immune system will be seminal for developing rational strategies for the prevention of COVID-19 infection, treatment of COVID-19associated diseases such as mucormycosis and postCOVID symptoms, and coping with the negative side effects of vaccines. Despite its important historical role, vitamin A has not attracted attention for the prophylaxis and treatment of COVID-19 during the pandemic. We think both vitamins A and D are important in regulating immunity as they act through retinoid signaling that orchestrates a proper immune response (Sarohan et al. 2021). Although some studies on vitamin D were carried out, these studies were not sufficient for the use of vitamins A and/or D against COVID-19 on a global scale. In addition, large-scale epidemiological studies have not been conducted to explain the geographical distribution of COVID-19, its relationship with comorbid diseases, demographic characteristics, and its relationship with nutritional status, and vitamin A and D deficiencies during the pandemic.

A. R. Sarohan et al. Conflicts of Interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Credit Authors Statement Aziz Rodan Sarohan wrote the first draft and discussed and finalized it throughout the process. Sait Edipsoy, Can Özlü, and Zeynep Gürsel Özkurt reviewed the draft and provided feedback. Ayça Nur Demir reviewed the draft, provided feedback, and revised the reference list. Osman Cen revised and rewrote the manuscript. Funding None

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166 income countries between 1991 and 2013: a pooled analysis of population-based surveys. Lancet Glob Health 3:e528–e536 Tanumihardjo SA (2011) Vitamin A: biomarkers of nutrition for development. Am J Clin Nutr 94:658s–665s Tepasse P-R, Vollenberg R, Fobker M, Kabar I, Schmidt H, Meier JA, Nowacki T, Hüsing-Kabar A (2021) Vitamin A plasma levels in COVID-19 patients: a prospective multicenter study and hypothesis. Nutrients 13:2173 Tian D, Sun Y, Xu H, Ye Q (2022) The emergence and epidemic characteristics of the highly mutated SARS-CoV-2 Omicron variant. J Med Virol 94: 2376–2383 Tielsch JM, Rahmathullah L, Katz J, Thulasiraj RD, Coles C, Sheeladevi S, Prakash K (2008) Maternal night blindness during pregnancy is associated with low birthweight, morbidity, and poor growth in South India. J Nutr 138:787–792 Timoneda J, Rodríguez-Fernández L, Zaragozá R, Marín MP, Cabezuelo MT, Torres L, Viña JR, Barber T (2018) Vitamin A deficiency and the lung. Nutrients 10:1132 Tomasa-Irriguible TM, Bielsa-Berrocal L, Bordejé-Laguna L, Tural-Llàcher C, Barallat J, ManresaDomínguez JM, Torán-Monserrat P (2021) Low levels of few micronutrients may impact COVID-19 disease progression: an observational study on the first wave. Metabolites 11:565 Toor R, Chana I (2022) Exploring diet associations with Covid-19 and other diseases: a network analysis-based approach. Med Biol Eng Comput 60:991–1013 Underwood BA (1994) Hypovitaminosis A: international programmatic issues. J Nutr 124:1467s–1472s Villar J, Merialdi M, Gülmezoglu AM, Abalos E, Carroli G, Kulier R, de Onis M (2003) Nutritional interventions during pregnancy for the prevention or treatment of maternal morbidity and preterm delivery: an overview of randomized controlled trials. J Nutr 133:1606s–1625s Vyas N, Kurian SJ, Bagchi D, Manu MK, Saravu K, Unnikrishnan MK, Mukhopadhyay C, Rao M, Miraj SS (2021) Vitamin D in prevention and treatment of

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Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 167–185 https://doi.org/10.1007/5584_2023_775 # The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 Published online: 28 May 2023

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput Screening for Precision Cancer Therapy Thudzelani Takalani Austin Malise, Ekene Emmanuel Nweke, Mutsa M. Takundwa, Pascaline Fonteh Fru, and Deepak B. Thimiri Govinda Raj Abstract

In the past few years, development of approved drug candidates has improved the disease management of multiple myeloma (MM). However, due to drug resistance, some of the patients do not respond positively, while some of the patients acquire drug resistance, thereby these patients eventually relapse. Hence, there are no other therapeutic

T. T. A. Malise, E. E. Nweke, and P. F. Fru Department of Surgery, University of the Witwatersrand, Johannesburg, South Africa M. M. Takundwa Synthetic Nanobiotechnology and Biomachines, Synthetic Biology and Precision Medicine Centre, NextGeneration Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa D. B. Thimiri Govinda Raj (✉) Department of Surgery, University of the Witwatersrand, Johannesburg, South Africa Synthetic Nanobiotechnology and Biomachines, Synthetic Biology and Precision Medicine Centre, NextGeneration Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa

options for multiple myeloma patients. Therefore, this necessitates a precision-based approach to multiple myeloma therapy. The use of patient’s samples to test drug sensitivity to increase efficacy and reduce treatmentrelated toxicities is the goal of functional precision medicine. Platforms such as highthroughput-based drug repurposing technology can be used to select effective single drug and drug combinations based on the efficacy and toxicity studies within a time frame of couple of weeks. In this article, we describe the clinical and cytogenetic features of MM. We highlight the various treatment strategies and elaborate on the role of highthroughput screening platforms in a precisionbased approach towards clinical treatment. Keywords

Drug screening · Drug sensitivity · Heterogeneity · Multiple myeloma · Precision medicine · Treatment strategies

Biotechnology Innovation Centre, Rhodes University, Grahamstown, South Africa

Abbreviations

Faculty of Medicine, University of Pretoria, Pretoria, South Africa e-mail: [email protected]

BM BMSC

Bone marrow BM-derived stem/stromal cells 167

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CAM-DR CDKN2A CRAB DAPK EC ECM FISH HATs HDACs HTS IGF-1 JAK/ STAT LDH MAPK MGMT MGUS MM mTOR NF-κB OB OC OS PDGF Ras SOCS-1 ß2M TERT TIMP3 VEGF VLA-4

1

Cell adhesion-mediated drug resistance Cyclin-dependent kinase inhibitors (CDKN2A, CDKN2C) Calcium, Renal, Anaemia, and Bone E-cadherin death-associated protein kinase Endothelial cells Extracellular matrix Fluorescence in situ hybridisation Histone acetylases Histone deacetylases High-throughput screening Insulin-like growth factor 1 Janus kinase, signal transducer and activator of transcription proteins Lactate dehydrogenase Mitogen-activated protein kinase O6-Methylguanine-DNA methyltransferase Monoclonal gammopathy of undetermined significance Multiple myeloma Mammalian target of rapamycin Nuclear factor kappa beta Osteoblasts Osteoclasts Overall survival Platelet-derived growth factor Rat sarcoma virus Suppressor of cytokine signalling Beta 2-microglobulin Telomerase reverse transcriptase Tissue inhibitor of metalloproteinase 3 Vascular endothelial growth factor Very late antigen 4

Introduction

Multiple myeloma (MM) is a plasma cell-based malignant disorder in the bone marrow (BM), characterised by the accumulation of monoclonal protein in serum and urine (Gulla and Anderson 2020; Landgren et al. 2009). The Global Cancer

Observatory (GLOBOCAN) reported 117,077 (66%) deaths from 176,404 MM cases worldwide in 2020 (The Global Cancer Observatory 2020). Data from the United States show an estimated death of 12,800 MM patients and account for 2.1% of all types of cancer deaths in 2020 (Padala et al. 2021). The median age of MM patients in the United States is 69, with more than 60% of MM are from patients older than 65 years (Padala et al. 2021). In 2010, the survival age range for older patients was 2 to 3 years, and for younger patients it was 5 to 6 years. Additionally, these survival ranges can depend on the stage of disease at diagnosis (Bladé et al. 2010; Padala et al. 2021). For a long time, MM patients have been treated with a first-line treatment chemotherapy combination of melphalan and prednisone, hoping to achieve remission (Gregory et al. 1992). Treatment options have since improved with inclusion of proteasome inhibitors, immunomodulatory drugs, and autologous stem cells transplantation; however, even with these options, most patients do not respond favourably to the treatment with most relapsing. Additionally, there are instances where the side effects of the drugs outweigh the benefits (Roussel et al. 2014). For example, most patients who had a high-dose therapy of lenalidomide, bortezomib, and dexamethasone combination supported by stem cell transplantation experienced a better response after induction, even though some common adverse events such as neuropathy, neutropenia, and thrombocytopenia can still be observed (Roussel et al. 2014). Drug regimen allocation to MM patients depends on drug availability, disease severity, age of the patient, and cytogenetics (Laudin et al. 2020; Mateo et al. 2005). If the treatment outcome is not favourable, patients are enrolled into clinical trials where treatment is in a randomisation format (Laudin et al. 2020; Mateo et al. 2005). As a result, there is an insufficient number of effective treatment options for MM patients capable of achieving a complete response and there is a critical need for a library of MM treatment data to reduce morbidity and mortality.

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput. . .

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Clinical and Pathological Characteristics of Multiple Myeloma

The first diversity of MM patients is observed on signs and symptoms, for example, most MM patients in the 1999 Moosa Patel study had bone pain (94.1%), followed by anaemia (81.2%), infections (59.8%), while 40.6% had plasmacytomas and 88.4% showed lytic bone lesions (Patel 1999). Multiple myeloma patients can also be asymptomatic with less bone pain and just headaches (Rankapole et al. 2011). Additional features include bone fractures, which require the use of an orthotic device to effectively mobilise, and the incidence of an extramedullary disease (Laudin et al. 2020). These findings show that MM patients are a diverse population that would require extensive strategies of management for all the ailments. Multiple myeloma is mainly characterised by an increase of clonal plasma cells in the BM (Matsue et al. 2017). Other diagnostic features include the detection of monoclonal protein in serum or urine (Reddy et al. 2021), and the evidence of end-organ damage, which includes hypercalcaemia, renal insufficiency, anaemia, and/or osteolytic bone lesions satisfying the CRAB criteria (Dinner et al. 2013). The CRAB criteria occurs because the neoplastic plasma cells of MM patients in the BM proliferate frequently and invade the adjacent bone, causing osteolytic lesions. Neoplastic plasma cell proliferation also interferes with the production of red blood cells causing anaemia (Kyle et al. 2003). The abundant production of the abnormal monoclonal proteins by MM cells instead of the normal proteins results in renal insufficiency and the occurrence of recurrent infections (Kyle et al. 2003). Multiple myeloma is preceded by two premalignant phases, the monoclonal gammopathy of undetermined significance (MGUS) and smouldering multiple myeloma (SMM) (Prideaux et al. 2014). During these stages, plasma cells experience sequential genetic and epigenetic events, and the BM also changes to create a conducive

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environment for MM cells to survive and proliferate (Giannakoulas et al. 2021). Accordingly, the patients in the first phase (MGUS) are asymptomatic, and their serum monoclonal protein levels and plasma cell clones in the BM are lower than 30 g/L and 10%, respectively. In contrast, SMM patients’ serum monoclonal protein levels and plasma cell clones are more than 30 g/L and 10%, respectively. To satisfy MM diagnosis, the patients must have a myeloma-related organ or tissue impairment (ROTI) and also have serum monoclonal protein levels and plasma cell clones at least >30 g/L and ≥10% in the BM, respectively (Chai et al. 2019; Matsue et al. 2017; Prideaux et al. 2014). Moreover, the clonal plasma cell percentage is also used during intensive care of MM patients, where a complete response is defined as 30% BM plasma cell infiltration equates to a shift from minor to major disease, a criterion which is used as a predictor of relapse in cases of treated MM (Štifter et al. 2010). Therefore, it is crucial to assess the patient’s clonal plasma cell percentages during treatment at specified time points to determine the effectiveness of the drug regimen. In addition to different levels of plasma cell infiltration in the BM, MM is characteristically heterogeneous and exhibits diverse genetic, molecular, and clinical features (Giannakoulas et al. 2021). The different attributes are used in risk assessments and have been adopted in the revised International Staging System (R-ISS) to classify MM disease into three stages using beta 2-microglobulin (ß2M) and albumin, lactate dehydrogenase (LDH) serum levels, patients cytogenetics, gene expression profiles, plasma cell proliferation, and the presence of extramedullary disease (Giannakoulas et al. 2021; Moser-Katz et al. 2021). Patients at stages I, II, and III had a 5-year overall survival (OS) of 82%, 62%, and 40%, respectively, showing that the level of risk determines the prognosis (Giri et al. 2020).

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Cytogenetics of Multiple Myeloma

Multiple myeloma cytogenetic aberrations are complex and crucial for determining disease severity and sensitivity to treatment. Newly diagnosed MM patients can be stratified into prognostic groups of genetic aberrations, which are attained using the standard cytogenetics and fluorescence in situ hybridisation (FISH) technique (Catamero 2018; Sawyer 2011). Multiple myeloma patients can be split into two main groups, the hyperdiploid and nonhyperdiploid based on their karyotype (Bianchi and Munshi 2015; de Smedt et al. 2018). The nonhyperdiploid karyotype group comprises the hypodiploid/pseudodiploid (which also includes the near-tetraploid karyotypes) (de Reave and Vanderkerken 2005). The hyperdiploid karyotype group is characterised by trisomies in odd chromosomes (3, 5, 7, 9, 11, 15, 19, and 21) (Bianchi and Munshi 2015) and is displayed in 50% to 60% of MM patients at diagnosis (de Smedt et al. 2018). The nonhyperdiploid karyotype chromosomal abnormalities include monosomy 13, 14, and 22; deletions of 1p, 12p, 16q, and 17p; and gains of 1p defects (van Wier et al. 2013). Nonhyperdiploid aberrations are observed in 40% to 50% of MM patients (Corre et al. 2015). Hyperdiploid karyotype has a better prognosis compared to the nonhyperdiploid karyotype group (Sawyer 2011). Multiple myeloma patients with t(4;14), t(14;16), and t(14;20) translocations, del(17p), are classified as high-risk disease and have a poor prognosis, in contrast, patients with t(11;14), t(6;14), and/or hyperdiploidy are classified as standard-risk disease and have a better prognosis (Catamero 2018; Diamantidis et al. 2022).

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Multiple Myeloma Clonal Expansion

Multiple myeloma evolves from the premalignant phases (Prideaux et al. 2014), at a rate of 1% per year from MGUS, and this goes with the idea that

genetic changes such as t(4;14), t(14;16), and 17p deletion start at the MGUS phase (López-Corral et al. 2011). López-Corral et al. (2011) assessed the incidence of genetic abnormalities in 565 patients, 90 MGUS, 102 SMM, and 373 MM, and they also observed the frequency of 13q, 17p deletions, 1q gains, t(11;14), t(4;14), and 13q deletions were all significantly less in MGUS compared to SMM and MM indicating that cytogenetic alteration accumulation is a necessary step during disease progression (LópezCorral et al. 2011). They also investigated if the accumulation of cytogenetic was related to the complications in MM. The authors demonstrated that the occurrence of t(14;16) translocation was associated with renal failure, and that, t(11;14) and t(6;14) were more common in patients with bone disease indicating that cytogenetics abnormalities are a good indicator of MM prognosis (Greenberg et al. 2014). Furthermore, there is also a difference in genetic aberrations observed at MM diagnosis and relapse. At relapse, the clones can either gain or lose chromosomal/genetic aberrations constituting intra-tumoural heterogeneity depending on the treatment the patient has been subjected to (Corre et al. 2015). In patients who received 4 cycles of either immunomodulatory or proteasome inhibitors-based treatment, 13q deletion and 1q21 gain tended to appear earlier, followed by a later attainment of 16q and 17p deletion. The appearance of 13q deletion was associated with worse survival than 1q21 gain (Yan et al. 2022). However, the prognostic value of 1q21 gain was variable at relapse due to changes in copy number and clone size. Patients who had an increase in copy number of 1q21 or who developed de novo 1q21 gain at relapse experienced the poorest outcome when compared to patients who had a reduced 1q21 copy number or those without or those who experienced no change to 1q21 gain (Yan et al. 2022). Genetic heterogeneity was further shown in MM patients who became double-refractory, triple-refractory, or quadruple-refractory to immunomodulatory and protease inhibitors at relapse (Giesen et al. 2022). When compared to newly diagnosed MM patients, the relapsed MM

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput. . .

patients showed more aberrations, including gain (1q) and deletions of 1p, 13q, and 17p, and a high frequency of functional small nucleotide variants per patient (Giesen et al. 2022). The mechanism in which genetic aberrations develop in MM cells was perfectly demonstrated by Shen et al. (2021) using a “bone chip” xenograft mouse model named “MM PrEDiCT” for MM progression through evolution and dissemination of clonal tumour cells (Shen et al. 2021). The “MM PrEDiCT” model allows the implantation, dissemination, and easy tracking of fluorescent-tagged or DNA-barcoded cells on SCID mice. After cell transplantation, SCID mice BM was colonised at 6 weeks and on the 8th week and host mice presented with limb paralysis, multiple skeletal lytic lesions, and a patchy distribution of fluorescent-tagged cells with some colours surpassing other colours. Upon disease progression, there was less colour diversity, showing that the most competent cell clones could outcompete other clones (Shen et al. 2021). Dominant clones were the same, in both left and right femur bones, and only differed when the host is changed (Shen et al. 2021). This shows that when provided with conditions mimicking the BM microenvironment, dominant MM cell clones will prevail and that each host micro-microenvironment is different and therefore contributes to heterogeneity.

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Genes Expression and Signalling

Some chromosome translocations may result in the overexpression of genes such as CCND1, CCND3, MAF, MAFB, WHSC1/MMSET, and FGFR3 via their juxtaposition to the immunoglobulin heavy chain (IgH) locus (Chapman et al. 2011). Other notable mutations occur in RAS family members (NRAS, KRAS, and BRAF), NFKB1, NFKB2, TRAF3, CHUK, and TP53, which can lead to the activation of signalling pathways (Giesen et al. 2022). In addition, during MM malignant progression, there is also MYC activation and loss-of-function mutations in the histone demethylase UTX/KDM6A (Chapman et al. 2011).

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In broad terms, pathways involved in MM progression include the p53 signalling, nuclear factor kappa beta (NF-κB) pathway, the cellcycle pathway, the p38 mitogen-activated protein kinase (MAPK) pathway, histone methyltransferase, the telomerase reverse transcriptase (TERT) pathway, Janus kinase, signal transducer and activator of transcription proteins (JAK/STAT), platelet-derived growth factor (PDGF) signalling pathways (Lam et al. 2018), rat sarcoma virus (Ras), mammalian target of rapamycin (mTOR), Notch-signalling pathway, vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) (Hou et al. 2019), and the cyclin-dependent kinases (CDKs), CDK1, and CDK2 (Lam et al. 2018). The functions of these pathways include tumour progression, survival, and escaping of tumour neutralising immune surveillance in the tumour microenvironment (Tai et al. 2018).

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Heterogeneity in Multiple Myeloma: The Role of Bone Marrow Microenvironment

The BM microenvironment provides the extracellular matrix (ECM) a layered structure that supports the myeloma cells (Moser-Katz et al. 2021). The ECM is made up of proteins such as fibronectin, type1 collagen, osteopontin (Hiasa et al. 2021), hyaluronan, and laminin (MoserKatz et al. 2021). Besides MM cells, the BM microenvironment also contains BM-derived stem/stromal cells (BMSCs), osteoclasts (OCs) (Hou et al. 2019), osteoblasts (OBs), adipocytes, vascular endothelial cells (ECs) (de Reave and Vanderkerken 2005), and immune cells (macrophages, neutrophils, natural killer (NK) cells, regulatory T cells (Tregs), etc.) (Giannakoulas et al. 2021) whose interactions are mediated by chemokines, cytokines, growth factors (Hou et al. 2019), receptors, and adhesion molecules (de Reave and Vanderkerken 2005). In the pathophysiology of MM, interactions between MM cells and the BM microenvironment are crucial. These components help with the survival of MM cells through various strategies

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including homing of MM cells to the BM, spreading via microvasculature to other sites of the BM, and secretion of growth factors resulting in differentiation; proliferation; drug resistance; osteoclastogenesis; inhibition of osteogenesis, angiogenesis, humoral and cellular immunodeficiency; and anaemia (de Reave and Vanderkerken 2005).

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Extracellular Matrix

The attraction of MM cells to the BM is mediated through the interaction of myeloma receptor CXCR4 with the chemokine stromal cell-derived factor 1 alpha (SDF1α) which results in migration towards stromal compartment of bone marrow (Moser-Katz et al. 2021), where they adhere to ECM proteins fibronectin and type I collagen (de Reave and Vanderkerken 2005), using the very late antigen 4 (VLA-4), an adhesion molecule expressed on MM cell surface (Ho et al. 2020). The VLA-4-fibronectin adherence results in the activation of NF-κB resulting in cell adhesion-mediated drug resistance (CAM-DR) and pro-survival signalling (Moser-Katz et al. 2021). Multiple myeloma cells also adhere to type I collagen via syndecan-1 or CD138, a heparan sulphate proteoglycan expressed on their surface (Moser-Katz et al. 2021) to induce the expression of matrix metalloproteinase 1 (MMP1), thereby promoting bone resorption, tumour invasion, angiogenesis, and ultimately MM cell survival (de Reave and Vanderkerken 2005). Additionally, MM cells adhere to the ECM via CD38 a hyaluronan receptor which is highly expressed in plasma cells but less in other lymphoid and myeloid cells (Moser-Katz et al. 2021).

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Bone Marrow-Derived Stromal Cells

Multiple myeloma cells utilise VLA-4 to adhere to vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule 1 (ICAM-1) on BMSCs (Ho et al. 2020). The adhesion of MM cells to BMSCs results in the secretion of

various growth and antiapoptotic factors, such as VEGF, IGF-1, basic fibroblast growth factor, angiopoietin 1, transforming-growth factor (TGF)-b, hepatocyte growth factor, stromal cellderived factor 1 (SDF-1), B cell-activating factor (BAFF), IL-21, IL-6, and IL-1 (Ho et al. 2020). The adhesion of MM cells to BMSCs and the secretion of growth and antiapoptotic factors lead to the activation of several pathways including the NF-κB, PI3K/Akt that mediate MM cell growth and survival (Hiasa et al. 2021).

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Cytokines

In MM, there are a number of cytokines which are crucial in proliferation, migration, and drug resistance, including IL-6, vascular endothelial growth factor (VEGF), tumour necrosis factor-α (TNF-α), BAFF, a proliferation inducing ligand (APRIL), colony stimulating factor-1 (CSF1), fibroblast growth factor-2 (FGF-2), angiopoietin-1, and others (Giannakoulas et al. 2021). Key cytokines like VEGF, IL-6, TNF-α, BAFF, and RANKL medium serum levels were shown to be higher in relapsed MM patients compared to newly diagnosed patients, indicating that they are crucial for MM progression (Jasrotia et al. 2020). Interleukin-6 is the most important cytokine in MM, since it has several functions including growth, survival, migration, invasion, angiogenesis, apoptosis inflammation, and drug resistance (Hou et al. 2019; Moser-Katz et al. 2021). It is produced by osteoblasts, monocytes, macrophages, and BMSCs (Andrews et al. 2013). For example, the secretion of IL-1ß, TGF-ß, TNF-α, bFGF, and VEGF by MM cells results in the induction of the secretion of IL-6 by the BMSCs (de Reave and Vanderkerken 2005). The binding of the secreted IL-6 to its receptor IL-6Ro on MM cells triggers the activation of the PI3K/Akt/mTOR and Ras/Raf/MEK/Erk signalling pathways and its growth factor role is through the phosphorylation of STAT3 via JAK1 (Hou et al. 2019). The phosphorylation of STAT3 results in the activation of Bcl-xL and myeloid cell factor-1 (mcl-1) which are crucial for MM survival (de Reave and Vanderkerken

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput. . .

2005). The activation of the phosphoinositol 3 kinase (PI3K)-protein kinase B (PkB/Akt) pathway by IL-6 provides an antiapoptotic activity and activation of Ras result in its translocation to the plasma membrane where it activates Raf, mitogen-activated protein kinase (MEKK), and MAPK, leading to increased proliferation of MM cells (Andrews et al. 2013). Furthermore, IL-6 promotes osteolysis (bone resorption) through the induction and production of RANKL, found on the surface of BMSCs and osteoblasts (Andrews et al. 2013). In addition, IL-6 may also contribute to immune dysfunction in MM (Prabhala et al. 2010). Tumour necrosis factor alpha is a selfregulating inflammatory cytokine that has a variety of biological functions, including angiogenesis, proliferation, immortalisation, and the induction of expression of adhesion molecules (Ho et al. 2020; Lemancewicz et al. 2013; Moser-Katz et al. 2021). The mechanism of TNF-α includes canonical NF-kB pathway activation and subsequent potent stimulation of autocrine IL-6 production (Ho et al. 2020). BAFF and APRIL, which are members of the TNF-α family (Bolkun et al. 2014), are highly expressed in MM cells as compared to normal plasma cells of the BM (Moser-Katz et al. 2021), and their expressions are associated with increased BM microvascular density (Bolkun et al. 2014). Furthermore, BAFF and APRIL were shown to contribute to a shorter progres sion-free survival and poor disease outcome (Lemancewicz et al. 2013). The APRIL protein, which is characteristically secreted without cell surface expression, binds to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) (Moreaux et al. 2004; MoserKatz et al. 2021). In contrast, BAFF is produced as both a membrane-bound and a proteolytically cleaved soluble protein and binds to BCMA, TACI, and a third receptor called BAFF-R (Moreaux et al. 2004). Even though there are minor differences between the two cytokines, they both activate the NF-κB, phosphatidylinositol-3 (PI-3) kinase/AKT, and MAPK kinase pathways in MM cells which result in promotion of cell

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growth, up-regulation of the Mcl-1 and Bcl-2 antiapoptotic proteins, and immunosuppression via programmed death-1, TGFß, and IL10 (Moreaux et al. 2004; Moser-Katz et al. 2021). The role of insulin growth factor in MM includes regulating the activities of proteasome and telomerase by binding to tyrosine kinase receptor IGF-1R and the priming of MM cells for response of action towards other cytokines and to produce pro-angiogenic cytokines (Moser-Katz et al. 2021). The mechanism of IGF-1 is through the activation of MAPK and PI3K/AKT signalling leads to Bcl-xL and Bcl-2like protein 11 (BCL2L11) or Bcl-2 interacting mediator of cell death (BIM) (Ho et al. 2020). Vascular endothelial growth factor, on the other hand, is one of the pro-angiogenic cytokines produced in MM by multiple cell types and is responsible for paracrine and autocrine growth of MM cells (Bolkun et al. 2014) and is key for angiogenesis and supports both microvascular endothelial cells and BMSCs through RAS, focal adhesion kinase, PI3K/AKT, MAPK, and STAT signalling (Ho et al. 2020). The mechanism of VEGF is thought to be a feedback loop in which MM cells stimulated by IL-6 secrete VEGF, which causes microvascular endothelial cells and BMSCs to secrete IL-6 (Paesler et al. 2012).

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Immunomodulatory Cells

Multiple myeloma progression is characterised by immune evasion, which occurs when dysfunctional effector lymphocytes, tumoureducated immunosuppressive cells, and soluble mediators work together to form a barrier to antimyeloma immunity (Nakamura et al. 2020). The macrophages whose population is the most abundant in the BM microenvironment have been shown to support MM cells (Kim et al. 2012). Macrophages induced an increase in the proliferation of MM cells, when co-cultured with BMSCs and showed a reduced proliferation when treated with IL-6 blocking antibody indicating that synergistic effect in the BM microenvironment is mediated by IL-6 (Kim et al.

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2012). The role of macrophages on MM cell growth was also examined on chemotherapytreated MM cells. Multiple myeloma cells co-cultured with macrophages resisted dexamethasone- and melphalan-induced apoptosis, to a greater extent with those co-cultured with tumour-derived macrophages than normal macrophages. Moreover, the co-culture of myeloma cells with tumour-derived macrophages also inhibited the activation and cleavage of caspase-3 and poly (ADP-ribose) polymerase PARP and maintained the levels of Bcl-xL and IL-6 (Zheng et al. 2009). Neutrophils isolated from MM patients express high levels of immunosuppressive molecule arginase-1 (Arg-1) as compared as those isolated from MGUS and healthy controls. Furthermore, they were also shown to have a reduced phagocytic activity and an immunosuppressive function on T lymphocytes (Parrinello et al. 2013). Eosinophils were shown to enhance the proliferation of MM cell lines and primary CD138+ MM cells and more when co-cultured in direct contact compared to on transwells (Wong et al. 2013).

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Epigenetics in Multiple Myeloma

Epigenetic alterations in tumours are much more frequent than the existing identified genetic aberrations, and these epigenetic variations are involved in signalling pathways for cell growth, proliferation, apoptosis, immune escape, energy metabolism disorders, and promotion of tumour inflammation (Liu et al. 2022). Similar to other cancers, the epigenetic machinery plays a crucial role in MM genomic instability and function (de Smedt et al. 2018). Epigenetic modifications include histone deacetylation/acetylation or DNA methylation (Liu et al. 2022). In addition, dynamic spatiotemporal positioning of nucleosomes, control of chromatin three-dimensional conformation and nuclear topology, control of non-coding RNA, microRNA, and enhancer RNA are all aspects of epigenetic regulation (Liu et al. 2022). Histone deacetylation/acetylation processes are controlled by the activity of histone acetylases

(HATs) and histone deacetylases (HDACs); therefore, the balance between these enzymes can alter the gene expression profile and some signalling pathways, including ERK (extracellular signal-regulated kinase) and Wnt (wingless/ Int-1), and can affect proteasomal degradation, influence protein kinase C activity, and change the DNA methylation status (Eckschlager et al. 2017). Increased histone acetylation causes decondensation of the chromatin, a process that can be reversed by HDACs (Eckschlager et al. 2017). Histone deacetylases reverse the decondensation by removing acetyl groups from protein tails causing the lysine residues to regain a positive charge and the resumption of the electrostatic contact with the DNA molecules resulting in the suppression of transcription (Garmpi et al. 2018). The acetylation process can also happen to non-histone proteins, altering numerous cellular processes (Eckschlager et al. 2017). Histone deacetylases regulate the functionality of various cytoplasmic proteins and transcription factors including tumour protein 53 (p53), RUNX3, signal transduction and activation of transcription 3 (STAT3), β-catenin, oestrogen receptor, avian myelocytomatosis viral oncogene homologue (Myc), erythroid Kruppel-like factor (EKLF), GATA family (GATA-binding factors), HIF-1alpha (hypoxia-inducible factor 1alpha), myogenic regulatory factor (MyoD), NF-κB, forkhead box P3 protein (Foxp3), E2F, GATA1, Bcl-6, HMG, HSP90, tubulin, ibortine, nuclear hormone receptors, and β-vaccine (Eckschlager et al. 2017; Garmpi et al. 2018). DNA methylation of the CpG dinucleotides in a gene’s promoter region prevents the binding of transcription factors resulting in reduced expression of genes situated near and far from the methylation site of a chromosome (de Reave and Vanderkerken 2005). Genomic instability in MM is caused by both hypomethylation and hypermethylation of intergenic regions, associated CpG islands of tumour suppressor genes, and microRNAs (Wang et al. 2017). Several cancer related genes such as cell-cycle regulators (p15, p16, and p18, p53, p73), tissue inhibitor of metalloproteinase 3 (TIMP3), E-cadherin death-associated protein kinase (DAPK), suppressor of cytokine

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput. . .

signalling (SOCS)-1, the oestrogen receptor and O6-methylguanine-DNA methyltransferase (MGMT), and cyclin-dependent kinase inhibitors (CDKN2A, CDKN2C) are all affected by the methylation process (de Reave and Vanderkerken 2005; de Smedt et al. 2018). Patients with p16 methylation were shown to have inferior clinical outcomes compared to those whose p16 is unmethylated (de Reave and Vanderkerken 2005).

morbidity, and increased mutational load at relapse and was followed by the introduction of cyclophosphamide, which has less myelotoxic effects (Medical Research Council 1971; Schjesvold and Oriol 2021). Treatment with cyclophosphamide is associated with high stem cell count irrespective of dosage; therefore, it is suitable for frail patients or those with impaired renal function (Zannetti et al. 2021).

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Multiple Myeloma Treatment Strategies

Since the 1960s, there has been an improvement in the strategy of managing MM (Kristinsson et al. 2007). Current strategies include radiation therapy, stem cell transplantation, cancer vaccines, and several compounds with distinctive mechanisms of action (Tai et al. 2018). In the subsequent sections, we discuss these methods in more detail.

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Alkylating Agents

Alkylating agents are compounds that react with the nucleophilic moieties of DNA molecules, resulting in the covalent transfer of an alkyl group; this causes the cross-linking of DNA strands, abnormal base pairing, or DNA strand breakage, and therefore the inhibition of cell division (Chiorcea-Paquim and Oliveira-Brett 2023; Cucchiara et al. 2022). Before the introduction of the alkylating agent, melphalan, in the 1960s, MM patients could not survive more than a year (Kumar et al. 2008). Melphalan increased the median OS to a year or two (Kristinsson et al. 2007). Treatment with melphalan induces cell death in highly proliferative cells by adding an alkyl group to DNA, causing strand linking and inhibiting DNA and RNA synthesis (Schjesvold and Oriol 2021). Melphalan has since been used in combination with prednisone or for mobilisation of autologous stem cells before collection and transplantation (Schjesvold and Oriol 2021). However, melphalan treatment is associated with prolonged recovery time,

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Glucocorticoids

Glucocorticoids are steroid hormones known to bind to cytosolic glucocorticoid receptors (GRs), causing it to translocate to the nucleus to modulate gene expression of genes such annexin I, MAPK phosphatase 1, nuclear factor κB, and activator protein-1 (AP-1), leading to the promotion of MM anti-inflammatory and immunosuppressive activities (Burwick and Sharma 2019). Prednisone was first used in combination with melphalan, providing proof that appropriate drug cocktails make are effective in for MM treatment (Costa et al. 1973). Currently, dexamethasone is used in combination with the immunomodulatory agent lenalidomide (Holstein and McCarthy 2017). Dexamethasone is used in combination with other drugs because it has been shown to have haemato-protective activity, prevents chemotherapy-induced toxicity, and has also been shown to have antiangiogenic effects, which helps to inhibit tumour growth (Gong et al. 2020). Glucocorticoids have been shown to improve the severity of immune effector cellassociated neurotoxicity syndrome (ICANS) and cytokine release syndrome (CRS) caused by CAR T cell immunotherapy (Wang et al. 2022).

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Antitumour Antibiotics (Anthracyclines)

Anthracyclines are antitumour antibiotics isolated from Streptomyces peucetius var. caesius (Sritharan and Sivalingam 2021). Doxorubicin (dox, Adriamycin) is a water-soluble anthracycline approved for MM treatment and its mechanism of

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action is through the inhibition of DNA, RNA protein synthesis (Plosker et al. 2008). The damage to proteins, lipids, and DNA is induced by the oxygen-derived free radicals produced by dox by using iron as a cofactor and the mitochondrial respiratory chain and they also act by DNA intercalation and through topoisomerase II poisoning (Martins-Teixeira and Carvalho 2020; Smith et al. 2010). In addition, dox also induces the increase in intracellular C6 ceramide levels that in turn aids the activation of AMP-activated protein kinase (AMPK), inhibition of mTORC1, chemosensitisation, and induction of cancer cell death (Sritharan and Sivalingam 2021). However, dox is mostly associated with adverse events, the prominent one being cardiotoxicity (Duggan and Keating 2011).

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Protease Inhibitors

The ubiquitin proteasome pathway (UPP) is key in maintaining the normal cellular homeostasis in eukaryotic cells through the degradation of proteins that control cell cycle, apoptosis, and DNA repair by eliminating dysfunctional or misfolded proteins via proteasome (Moreau et al. 2012). There is a high level of proteasome activity in MM cells as compared to normal cells, thereby making proteasome inhibition a crucial therapeutic strategy (Moreau et al. 2012). The introduction of bortezomib in the early 2000s improved the outcomes of MM patients (Kumar et al. 2008). Bortezomib inhibits the 26S proteasome directly by inhibiting the degradation of IκBα, an inhibitory protein that is constitutively bound to cytosolic NF-κB, thereby inhibiting the nuclear translocation and activation of NF-κB (Yang and Lin 2015). Bortezomib’s effects include the disruption of MM cells and BMSCs adhesion via IL-6 activated inhibition (Yang and Lin 2015). However, bortezomib causes peripheral neuropathy, and only a small proportion of MM patients benefit from its use (An et al. 2015). Carfilzomib was FDA approved in July 2012 to be a second-generation protease inhibitor for the treatment of MM patients who

have relapsed or are refractory, particularly those who received prior bortezomib and thalidomide/ lenalidomide treatment (Yang and Lin 2015). In addition, carfilzomib effects are irreversible, more selective, and have no off-target activity (Bai and Su 2021). Most importantly, carfilzomib causes limited neurotoxicity (Moreau et al. 2012). Ixazomib was introduced as the first oral protease inhibitor with reversible effects but more efficient for proteasome inhibition in MM than bortezomib (Bai and Su 2021). In addition, ixazomib has a shorter dissociation half-life, and it was shown to penetrate the cancerous tissue more efficiently (Moreau et al. 2012).

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Immunomodulatory Agents

Immunomodulatory drugs have a pleiotropy of properties against MM, including the ability to modulate host immune responses; influence cytokine secretion, angiogenesis, and induce inflammation (Sedlarikova et al. 2012). In 1999, thalidomide was introduced as the first immunomodulatory agent for MM treatment, and it exhibited improved response rate and progression-free survival of patients when used in combination with melphalan-prednisone therapy (Kumar et al. 2008). Thalidomide is a synthetic derivative of glutamic acid comprised of a chiral centre and functional (S)- and (R)- optical isomers with teratogenic and sedative effects, respectively (Sedlarikova et al. 2012). Thalidomide targets MM cells in the BM microenvironment by inhibiting TNF-α production and angiogenesis by blocking the angiogenic growth factors bFGF and VEGF (Yang and Lin 2015). Lenalidomide was FDA approved in 2006 as a secondary generation immunomodulatory agent for the treatment of relapsed/refractory MM, and it was further approved for use in combination with dexamethasone for the treatment of newly diagnosed MM patients (Holstein and McCarthy 2017). Pomalidomide was added in 2013 for the treatment of relapsed/refractory myeloma in for patients who had received at least two prior regimens (Holstein and McCarthy 2017).

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput. . .

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Monoclonal Antibodies

Monoclonal antibodies exert their action by binding to molecules expressed on MM cells and subsequently blocking the BM-MM cell interaction, producing a durable and less toxic response (Wudhikarn et al. 2020). The cluster of differentiation 38 (CD38) and signalling lymphocyte activation molecule family member 7 (SLAMF7) molecules are highly expressed on the surface of MM plasma cells, making them suitable for the development of cell surface targeted therapy (Romano et al. 2021). Daratumumab monoclonal antibody for MM treatment was approved in the United States by the FDA and by the European Medicines Agency (EMA) for Europe in 2015 and 2016, respectively (Federico et al. 2021). Daratumumab targets CD38 and induces an antitumour effect via several mechanisms, which include complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis, through the activation of complement proteins, NK cells, and macrophages, respectively (Federico et al. 2021). Isatuximab is another CD38 monoclonal antibody targeting CD38 that is used together with pomalidomide and dexamethasone as drug combinations, for the MM patient treatments after at least two line of treatments (Romano et al. 2021). The advantages of isatuximab are that it is more epitope specific and can also induce direct apoptosis without cross-linking (Wudhikarn et al. 2020). Elotuzumab, on the other hand, targets SLAMF7 and exerts the antimyeloma activity primarily via NK-mediated antibody-dependent cellular cytotoxicity through both direct activation and engagement of NK cells (Wudhikarn et al. 2020).

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Histone Deacetylase Inhibitors

Epigenetic regulation of gene expression is crucial for normal cell function. In cancer, there are aberrations in histone deacetylases (HDACs) (Garmpi et al. 2018), making them important therapeutic targets (Hideshima et al. 2011). Histone deacetylase inhibitors (HDACIs) reverse the effects of HDACS by inducing cell-cycle arrest,

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differentiation, and death, as well as reducing angiogenesis and modulating immune responses (Garmpi et al. 2018). For MM treatment, HDACIs such as panobinostat and vorinostat were shown to inhibit cell growth and induce apoptosis when used alone and in combination with bortezomib by interfering with protein degradation and preventing MM cells from interacting with the tumour microenvironment (Hideshima et al. 2011). The disadvantage of using HDACIs is that HDACs could contribute to cancer through other mechanisms other than overexpression, thus may not be effective (Garmpi et al. 2018).

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Stem Cell Transplantation

Stem cell transplantation after treatment with a high-dose-melphalan was first reported in 1996 (Kumar et al. 2008). The transplantation of stem cells helps in salvaging the patient after the increase of melphalan dose (Child et al. 2003). Patients receiving high-dose-melphalan plus stem cell transplantations have a good prognosis compared to those on standard therapy, though they are still at risk of infection due to the high doses of melphalan administered (Child et al. 2003). Double transplantation improved OS relapsefree survival and event-free survival values compared to single transplantation (Attal et al. 2003). However, not everyone can benefit from stem cell transplantation, since eligibility can be affected by the age and performance status of the patient (Attal et al. 2003). Another therapeutic strategy that affects autologous stem cell collection is radiotherapy (Damron et al. 2021). Radiotherapy in MM is used as a curative treatment for plasmacytomas and as palliation for local symptoms due to certain bone or extramedullary lesions (Momm et al. 2020).

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Cancer Vaccines

Since BM-MM interactions create an immunosuppressive microenvironment, the cancer vaccine treatment approach aims to stimulate the endogenous antimyeloma T cell responses by

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introducing MM antigens (Garfall and Stadtmauer 2016). Because MM resides primarily in the BM, where it is difficult to traffic cellular therapies to their targets, dendritic cells (DCs) augmented by differentiation/activation ex vivo before infusion for antigen presentation to elicit a cytotoxic T lymphocyte response are utilised (Garfall and Stadtmauer 2016). Various MM antigens that can effectively induce an immune response have been explored, and the importance of selecting suitable antigens for this purpose was observed by Lu et al. (2017). The study investigated whether multi-epitome peptide comprised of the MM special protein (MMSA-1) and Dickkopf-1 (DKK1) could induce an antitumour immune response (Lu et al. 2017). The MMSA-1 protein is predominantly distributed on cell membranes and to a lesser extent in the cytoplasm, while DKK1 is a key regulator of myeloma bone via the Wnt signalling pathway. The vaccine significantly alleviated bone destruction in MM and increased the frequencies of CD4+ and CD8+ T cells in the blood of mice, indicating that this vaccine elicits MM antitumour immune response (Lu et al. 2017). However, this process of dendritic cell generation would be timeconsuming and expensive. Moreover, good manufacturing practice protocols and quality assurance measures of ex vivo DCs are yet to be established (Verheye et al. 2022).

used to generate CAR T cells, starting with T cells selection, followed by stimulation, genetic modification, and expansion for infusion into the patient (Panch et al. 2019). CAR T cell therapy targets markers expressed on cancer cells, and for MM treatment, investigations have been directed on targeting BCMA, CD138, CD38, CD19, signalling lymphocyte activation molecule (SLAM) or CS1, κ light chain, GPRC5D, and NKG2D (Sidana and Shah 2019). The limitation to the use of autologous peripheral blood mononuclear cells is because the patient’s age, prior treatment regimens, and the degree of the disease can all have an impact on the quality of patient-derived T cells (Sommer et al. 2019). It might be advisable to isolate the T cells earlier before relapse (Battram et al. 2022). Nevertheless, there are still logistics problems in manufacturing and storing the CAR T cells (Panch et al. 2019; Sommer et al. 2019). In addition, individuals receiving CAR T cell immunotherapy experience side effects such as CRS, ICANS, cytopenias, tumour lysis syndrome, hypogammaglobulinaemia, hepatotoxicity, anaemia, and infections (Javed et al. 2020; Kambhampati et al. 2022).

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Multiple myeloma patients exhibit diverse cytogenetic abnormalities and distinct clinical and pathological characteristics, considering that clonal heterogeneity and evolution hints towards a precision-based approach to treatment. Precision medicine is an approach where a patient’s clinical and molecular characteristics can be used to determine treatment and management of a disease. Precision-based approaches can ensure that effective drugs and drug combinations can be identified for a patient (Nweke and Thimiri Govinda Raj 2021). Over recent years, there are growing interests in the evaluation of drugs that could be effective for patients using screening platforms. High-throughput screening (HTS) is a procedure of assaying many drug candidates using

CAR T Cell Therapy

The utilisation of the patient’s own immune system is one recent method for treating cancer cells. Examples of these techniques include utilising bispecific T cell-engaging antibodies, chimeric antigen receptor (CAR) T or CAR NK cells, or T cell or macrophage checkpoint inhibitors (Daver 2020). The CAR T cells are being investigated for the treatment of MM because they have demonstrated promise in treating haematological malignancies (Sidana and Shah 2019), and they have since been FDA approved in 2017 for cancer treatment (Panch et al. 2019). Normally, the patient’s peripheral blood mononuclear cells obtained by leukapheresis are

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Precision Medicine Approach: The Role of High-Throughput Screening

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miniaturised in vitro assays capable of identifying compounds that modulate biological targets of interest (Armstrong 1999; Pereira and Williams 2007). The HTS technique can be performed in 96-well, 384-well, or 1,536-well microplates, thereby, increasing the turnover and minimising the cost. Additionally, the HTS procedure uses the common assay types, including ELISA, CellTiter-Glo® luminescent cell viability, CellTox™ green cytotoxicity assays, or PrestoBlue® viability assay (Armstrong 1999). Examples of cancer drugs discovered from HTS include tyrosine kinase targets, such as Gefitinib (Iressa; AstraZeneca), Erlotinib (Tarceva, Roche), Sorafenib (Nexavar; Bayer/ Onyx Pharmaceuticals), Dasatinib (Sprycel; Bristol-Meyers Squibb), and Lapatinib (Tykerb; GlaxoSmithKline) (MacArron et al. 2011). In HTS, MM cells are treated with various drugs at different concentrations in microplates and cell viability readouts for drug sensitivity obtainable in 24–72 h by using a variety of cellbased assays, such as CellTiter-Glo® luminescent cell viability assay or CellTox™ green cytotoxicity assay or PrestoBlue® viability assay (Fig. 1) (Meurice et al. 2017; Thimiri Govinda Raj et al. 2018a, b). Drug sensitivity for each patient

sample can therefore be calculated by using the drug sensitivity score (DSS) method of calculation, an improved “Area Under the Curve (AUC)”. The drug sensitivity score integrates multiple dose-response parameters for each drug to obtain drug efficacy and response data across patients’ samples, using the IC50 value (half maximal inhibitory concentration), slope, and AUC (Majumder et al. 2017). A selective DSS (sDSS) can then be created for each drug by comparing the DSS values of MM patients to healthy controls (Thimiri Govinda Raj et al. 2018a, b). Thus, with HTS, more drugs can be tested against MM patient samples, consequently circumventing the need to carry out randomisation studies. A HTS platform can be used to optimise treatment options for multiple myeloma patients. Multiple myeloma patients can therefore be clustered based on their varied drug sensitivities. To obtain optimal treatment options for individual patients, Majumder et al. (2017) conducted a drug sensitivity and resistance study using 50 MM patient samples expressing CD138+ against 308 drugs at the clinically relevant range of 1-10,000 nM in 384-well drug plates. The selected drugs were the approved oncology

Fig. 1 Workflow for precision-based high-throughput screening platform. Patient samples are collected either from the bone marrow or whole blood. Mononuclear cells are then isolated by gradient separation and cultured for 1–3 days on precoated drug compounds microplates.

After 1–3 days, cell viability readouts are obtained using CellTiter-Glo® or PrestoBlue® viability assay and results analysed to get drug sensitivities. Potent drugs are then recommended for treatment

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drugs and novel agents meant to target multiple signalling pathways, including protease inhibitors, IMIDs, glucocorticoids, HDAC inhibitors, BCL2 inhibitors, PI3k-AKT-mTOR inhibitors, IGF1R inhibitors, MEK/ERK inhibitors, HSP90 inhibitors, CDK inhibitors, rapalogs, and others (Majumder et al. 2017). This allowed the authors to group each patient’s samples into four (I-IV) groups based on drug sensitivity scores. Patient samples in group I (n = 16) had massive sensitivities, followed by group II (n = 13), which had moderate sensitivities, and those in group III (n = 18) were insensitive to several drugs, and the fourth group (IV) samples (n = 3) were insensitive to almost all the drugs (Majumder et al. 2017). The authors also investigated the correlation between drug response profiles and karyotypes. Most patients with intermediate-risk t(4;14) cytogenic abnormality were in group II, and to a less extent in group III. Whereas those with the high-risk del (17p) cytogenic abnormality were predominant in groups III and IV (Majumder et al. 2017). The results show the heterogeneity of MM patient samples, and that prior drug sensitivity is required before subjecting the patients to a particular drug regimen. One study sought to establish the feasibility of HTS as a precision medicine tool for informing treatment decisions in real time. A total of 177 compounds, including both FDA-approved and investigational drug compounds, were screened on 16 patient samples taken from MM patients, who had, on average, received six lines of prior medication. The median turnaround time to get drug screening results was 5 days. The assay-guided therapy was administered in 13 of the 16 patients and attained a 46% overall response rate (Coffey et al. 2021). High-throughput screening platforms can also be beneficial in identifying effective synergistic drugs for combinatorial treatment. A recent study used a HTS platform to first identify a potent drug from single drug treatments and used it as a priming drug for a double- or triple-drug combination treatment screening. Carfilzomib, bortezomib, ixazomib, and panobinostat were found to be the most potent in single treatments, and in double combination treatments bortezomib-dexamethasone, panobinostat-

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melflufen, and carfilzomib-dexamethasone were more potent. Efficacy was widespread in tripledrug combination treatments bortezomib-dexamethasone-lenalidomide, bortezomib-dexamethasone-pomalidomide, dexamethasone-bortezomibselinexor, bortezomib-prednisolone-melphalan, and carfilzomib-dexamethasone-panobinostat were more potent (Giliberto et al. 2022).

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Future Perspectives and Conclusion

Over the years, the different treatment strategies discussed in this article have improved outcomes for MM patients. A study found that relative survival ratios have increased steadily from 1973 to 2003 (Kristinsson et al. 2007). There has also been an uptake in the utilisation of double- and triple-drug combinations, which have slightly improved patient outcomes. The use of proteasome inhibitors and immunomodulatory drugs supported by stem cell transplantation can enable improved leukemia treatment (Roussel et al. 2014). Taken together, these strategies still fail to significantly improve patient outcomes and quality of life. Some of the treated patients do not respond well, eventually develop resistance and the drugs themselves could be toxic. One major reason for the lack of favourable response to treatment is heterogeneity characteristic of MM. In this regard, a precision-based approach using platforms such as HTS could help in identifying optimal drug combinations. Authors Contribution Thudzelani Takalani Austin Malise wrote the manuscript together with the inputs of all authors, Ekene Emmanuel Nweke, Mutsa M Takundwa, Pascaline N Fru, and Deepak B. Thimiri Govinda Raj, revised, and approved the final version. Acknowledgements Thudzelani Takalani Austin Malise is funded by National Research Foundation (NRF) PhD Fellowship of South Africa. Deepak B. Thimiri Govinda Raj is funded by CSIR Strategic Initiative funding, NRF Competitive Grant, SAMRC SIR Grant, and ICGEB Early Career Grant. Mutsa M Takundwa was funded by National Research Foundation (NRF) Innovation Fellowship of South Africa.

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput. . .

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Adv Exp Med Biol - Cell Biology and Translational Medicine (2023) 20: 187–189 https://doi.org/10.1007/978-3-031-41688-0 # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

Index

A Acoustic force spectroscopy (AFS), 109–117 Acoustic radiation force, 111–113 Acoustic standing waves, 110, 111 Addis, R.C., 7, 9 Adhesion kinetics, 116 Adhesion strength, 116–117 Alevizos, I., 60, 61 Al-Gharaibeh, A., 28 Almansoori, A., 55–64 Almansoori, A.A., 119–128 Angiogenesis, 75, 76, 79, 135–138, 142, 172, 173, 176, 177 Arber, C., 28 Argenta, L.C., 132, 138 Arslan, U., 30 Aslan, A., 19–44 Assadian, O., 142 Azuma, R., 131–145 B Barczewska, M., 30 Bektik, E., 8, 12 Berry, J.D., 30 Biermann, N., 142 Biofilm, 141, 142 Biomechanics, 109–117 Blackburn, J.H., 143 Black fungus, 153–162 Boido, M., 25 Bonfield, T.L., 26 Boone, D., 141 Borthakur, A., 1–15 Boyer, J.G., 24 Bushara, K.O., 122, 124 C Cancer, 56, 57, 62, 71–81, 94–95, 97–98, 122, 139, 167–180 Cancer stem cells (CSCs), 69–81 Cao, N., 12 Cao, U.M.N., 55–64, 119–128 Carbone, A., 26

Cardiomyocytes, 2–15 Cardiovascular diseases (CVDs), 2, 126, 161 Caveolae, 89, 92, 93, 95–98 Cell therapy, 2, 13, 22, 33, 35, 44, 56, 57, 77, 78, 178 Cen, O., 153–162 Chal, J., 27 Chan-Il, C., 25 Chan, S.S., 27 Christoforou, N., 12 Cizmecioglu, O., 87–99 Clustered regularly interspaced short palindromic repeat (CRISPR), 32, 33, 39, 40, 42, 43 Conjunctivitis, 155, 158, 160 Conventional stem cell therapies, 22, 23, 30, 72, 77, 79 COVID-19, 57, 153–162 D Dai, A., 46 Delli Carri, A., 28 Demir, A.N., 153–162 Di Pasquale, G., 58 Direct cardiac reprogramming, 1–15 Drug screening, 180 Drug sensitivity, 179, 180 Drug therapeutics, 119–128 E Edipsoy, S., 153–162 Efe, J.A., 7, 9 Elbaz, E.M., 27 El Helou, G., 62 Epithelial-mesenchymal transition (EMT), 71–74, 76, 81, 91, 139, 140, 142 F Ferreira, J.N.A., 60 Fink, K.D., 27 Forostyak, S., 25 Fru, P.F., 167–180 Fu, J.-D., 11, 12 Fujikura, Y., 23 Fujiwara, T., 141

187

188 G Gelati, M., 31 Gene therapy, 22, 33, 36, 41, 44, 56, 57, 61–62, 64 Genetic disease, 19–44, 56 Gene transfer, 36, 37, 57–62 Genome editing tools, 21, 32, 40, 44 Guo, Y., 8 H Hai, B., 60 Haridhasapavalan, K.K., 1–15 Hariharan, A., 55–64, 119–128 Head and neck radiotherapy, 62, 122 Heterogeneity, 71, 74–76, 78, 79, 81, 170, 171, 178, 180 Hu, C., 141 Hu, L., 60 I Ieda, M., 7, 9, 11 Ifkovits, J.L., 7, 9 Integrative and non-integrative approaches, 6, 13, 41 Intraductal, 119–128 Intraglandular, 119–128 Ishihara, M., 131–145 Isik, O.A., 87–99 Isomi, M., 8 Iwatani, S., 25 J Jayawardena, T.M., 7, 9 Jeon, I., 28 Jiang, Y., 26 K Kalkan, R., 69–81 Kamsma, D., 116 Katzengold, R., 136 Keratitis, 155, 158–160 Kim, H.S., 25 Kim, J.W., 123 Kisby, T., 8, 10 Kushibiki, T., 131–145 Kuzma-Kozakiewicz, M., 31 L Lab-on-a-chip, 110 Lai, Z., 60 Lee, K., 7 Levy, S., 30 Lim, C.K., 8 Lin, C., 23 Lin, Y.T., 26 Lipid rafts, 87–99 Liu, L., 139 Liu, W., 140 Lombaert, I.M.A., 60

Index López-Corral, L., 170 Lu, C., 178 M MacKenzie, T.C., 31 Majumder, M.M., 179 Ma, L., 27 Malise, T.T.A., 167–180 Mangunsong, C., 30 Marconi, S., 25 Martinac, B., 109–117 Mastrangeli, A., 61 Mathison, M., 7 Mayumi, Y., 131–145 Ma, Z., 137 Mechanobiology, 110, 117 Mechanotransduction, 110, 135, 136, 139, 141 Microdeformational wound therapy (MDWT), 132 Microfluidic, 110–115 Miyamoto, K., 8, 12 Mohamed, T.M., 7 Mohseni, R., 25, 30 Moraes, L., 26 Morykwas, M.J., 132, 133, 138, 141 Mouës, C.M., 142 Mou, H., 29 Mucormycosis, 153–162 Multiple myeloma (MM), 167–180 Muraoka, N., 7, 9, 12 Muthumariappan, S., 126 N Nabavi, S.M., 30 Nam, Y.-J., 7, 11, 12 Ngo, Q.D., 141 Nguyen, A., 117 Night blindness, 155, 159 Nitahara-Kasahara, Y., 24 Nizzardo, M., 29 Nweke, E.E., 167–180 O Osum, M., 69–81 Özkurt, Z.G., 153–162 Özlü, C., 153–162 Özmert, E., 30 P Pal, P.K., 122 Paoletti, C., 12 Paracchini, V., 26 Patel, A., 30 Patel, V., 7 Peranteau, W.H., 27 Petrou, P., 30 Piñol-Jurado, P., 23

Index Plasma membrane compartmentalization, 88, 93, 96 Precision medicine, 178–180 Protze, S., 7 R Radiation damage, 62, 64 Riordan, N., 30 Romanov, V., 109–117 Rossignol, J., 26, 27 Rouger, K., 23 S Sakai-Takemura, F., 28 Salivary gland, 55–64, 119–128 Salivary gland regeneration, 62–64, 124 Salivary hypofunction, 122, 123 Sarohan, A.R., 153–162 Saxena, V., 136 Scherer, S.S., 133 Shang, Y.C., 24 Shaw, S.W., 25 Sherif, H., 122 Siemionow, M., 24 Signal transduction, 88, 89, 91, 95, 96, 174 Silvani, G., 109–117 Singh, V.P., 8, 12, 13 Single biomolecule, 110 Sironi, F., 25 Sjogren’s syndrome (SS), 58, 60, 62, 63, 125–127 Sogorski, A., 138 So, J.I., 125 Song, K., 7 Staff, N.P., 30 Sun, H., 25 Surgical site infection (SSI), 143 Sutton, M.T., 26 Sykova, E., 30 T Taetzsch, T., 24 Takundwa, M.M., 167–180 Talkhabi, M., 7 Testa, G., 8 Teymoortash, A., 122 Therapeutic targeting, 72, 77–81 Thimiri Govinda Raj, D.B., 167–180 Thummer, R.P., 1–15 Tian, J., 7 Topical negative pressure (TNP), 132 Toume, S., 136 Transcription activator-like effector nucleases (TALENs), 29, 32, 37, 39–43 Tran, S.D., 55–64, 119–128 Treatment, 2, 20, 56, 71, 95, 116, 120, 132, 154, 168 Treatment strategies, 167–180

189 Tsuchiya, M., 131–145 Tuekprakhon, A., 30 U Upadhyay, A., 55–64, 119–128 V Vacuum sealing drainage (VSD), 132 Van Rosmalen, M.G., 116 Veerasubramanian, P.K., 136 Verma, M., 24 Violatto, M.B., 25 Viscoelasticity, 113–115, 117 Vitamin A deficiency, 153–162 W Wackenfors, A., 138 Wada, R., 11, 12 Wang Feige, 23 Wang, G.Q., 141 Wang, H., 7, 10 Wang, J., 8, 13, 64 Wang, R., 141 Wang, Z., 59–61, 64 Weiss, J.N., 30 Wilkes, R.P., 138 Wound healing, 132–136, 138, 139, 141 Wu, C., 60 Wu, J.P., 141 X Xerophthalmia, 155, 158–160 Xerostomia, 62, 122, 124, 126 Y Yamashiro, T., 131–145 Yang, Z., 141 Yoon, Y., 28 Yoo, S.Y., 7, 10 Yuka, S.A., 19–44 Yu-Taeger, L., 27 Z Zeng, M., 60, 63 Zhang, H., 23 Zhang, Z., 8 Zhao, H., 8 Zhao, Y., 7, 9 Zhou, C., 25 Zhou, H., 7 Zhou, Y., 7 Zhu, J., 140 Zinc-finger nucleases (ZFN), 29, 37, 40, 41 Zou, X., 23 Zulueta, A., 26